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REVIEW ARTICLE
Molecular mechanisms of the PRL phosphatasesPablo Rios, Xun Li and Maja Kohn
European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany
Keywords
cancer; cell signalling; dual specificity
phosphatases; metastasis; phosphatase of
regenerating liver; protein phosphatases
Correspondence
M. Kohn, European Molecular Biology
Laboratory, Genome Biology Unit,
Meyerhofstrasse 1, 69117 Heidelberg,
Germany
Fax: +49 6221 387518
Tel: +49 6221 3878544
E-mail: koehn@embl.de
(Received 8 January 2012, revised 20
February 2012, accepted 9 March 2012)
doi:10.1111/j.1742-4658.2012.08565.x
The phosphatases of regenerating liver (PRLs) are an intriguing family of
dual specificity phosphatases due to their oncogenicity. The three members
are small, single domain enzymes. We provide an overview of the phospha-
tases of regenerating liver, compare them to related phosphatases, and
review recent reports about each phosphatase. Finally, we discuss similari-
ties and differences between the phosphatases of regenerating liver, focus-
ing on their molecular mechanisms and signalling pathways.
Common features of the PRLs
Ever since phosphatase of regenerating liver (PRL)-3
was found to be overexpressed in liver metastatic tissue
originating from colon cancer, but not in normal colon
tissue nor in the primary tumour [1], the PRL family
of phosphatases has received much attention. There is
strong evidence suggesting that not only PRL-3, but
also PRL-1 and PRL-2 are oncogenes and, as such,
belong to the few phosphatases that lead to the devel-
opment of cancer [2–5]. The PRLs promote cell prolif-
eration, migration, invasion, tumour growth and
metastasis [4,6–15], and these are recognized driving
forces behind their oncogenicity. The underlying
molecular mechanisms still remain undetermined,
although progress has been made in understanding the
proteins and pathways involved [2,3].
Rat PRL-1 phosphatase was the first member of the
PRL family to be discovered, and was found as an
immediate early gene induced in rat regenerating liver
and mitogen-stimulated cells, and to be constitutively
expressed in insulin-treated rat hepatoma H35 cells
[16,17]. Subsequently, human PRL-2 was identified in
a genetic study [18] and, together with human PRL-1,
in a prenylation screen as farnesylated proteins in vitro
through a C-terminal CAAX motif (where C is the
Abbreviations
AML, acute myeloid leukaemia; ATF, activated transcription factor; bGGT II, b-subunit of Rab geranylgeranyltransferase II; CDC14, cell
division cycle 14 homologue; CDK2, cyclin-dependent kinase 2; Csk, C-terminal Src kinase; EF-2, elongation factor 2; EMT, epithelial–
mesenchymal transition; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FKBP38, FK506-binding protein 38; GTPase,
guanosine triphosphate phosphohydrolase; KAP, kinase-associated phosphatase; MDM2, mouse double minute 2; MEF, mouse embryonic
fibroblast; MEF2C, myocyte enhancer factor 2C; MEKK1, mitogen-activated protein ⁄ extracellular signal-regulated kinase kinase kinase 1;
MMP, matrix metalloproteinase; OMFP, ortho-methylfluorescein phosphate; PCBP1, polyC-RNA-binding protein 1; PI3K, phosphatidylinositol
3-kinase; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PIRH2, protein with a RING-H2 domain; pNPP, para-nitrophenyl phosphate;
PRL, phosphatase of regenerating liver; PTEN, phosphatase and tensin homologue; PTP, protein tyrosine phosphatase; PTPMT1, protein
tyrosine phosphatase mitochondrial 1; rhoGAP, Rho-GTPase-activating protein; SH3, SRC homology 3 domain; siRNA, small interfering RNA;
Src, sarcoma; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; VHR, vaccinia H1-related phosphatase.
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 1
cysteine that is prenylated, A is an aliphatic amino
acid and X is any amino acid) [6]. In addition, the lat-
ter study already recognized the oncogenic potential of
these phosphatases. Finally, mouse PRL-2 and PRL-3
were cloned and analyzed with respect to their
sequence similarity to other phosphatases and the
expression pattern in mice [19]. Although the amino
acid identities of the PRLs are low compared to other
phosphatases, they are very high between the three
PRLs: 87% between PRL-1 and PRL-2; 79% between
PRL-1 and PRL-3; and 76% between PRL-2 and
PRL-3 in humans [2,3,20] (Fig. 1).
PRLs are classified into the family of dual specificity
phosphatases (DSP) (also called vaccinia H1-like phos-
phatases) [21], which is a subgroup of the class I pro-
tein tyrosine phosphatase family defined by the
conserved active site p-loop sequence HC(X)5R[S ⁄T][22]. PRLs are relatively small proteins of approxi-
mately 20 kDa. They do not have regulatory domains,
although they contain a variety of intrinsic regulatory
elements [2,3]. PRLs are the only phosphatases of the
protein tyrosine phosphatase (PTP) superfamily that
carry the aforementioned CAAX motif and are
farnesylated in vivo [23–25], and they can also be gera-
nylgeranylated in vitro [6,23,26]. However, reports
about PRL-3 geranylgeranylation are contradictory
[23,26]. Interestingly, the CAAX box is common
amongst human phosphatidylinositol-5-phosphatases
[27]. Prenylation of the PRL phosphatases localizes
them to the plasma membrane and intracellular
membranes in distinct punctate structures, which are
suggested to be the early endosome, and deletion of
the CAAX box or application of farnesyl transferase
inhibitors prevents membrane localization and
redirects the PRLs into the nucleus [10,23,28]. The cat-
alytic activity and farnesylation are necessary for the
cellular and tumour- and metastasis-related phenotypes
of the PRLs [8,28–30]. In addition, similar to members
of the Ras superfamily of guanosine triphosphate
phosphohydrolases (GTPases), the PRLs carry a
Fig. 1. Structure-based multiple sequence alignment of PRLs and the structurally most closely-related PTPs. Ci-VSP is included as a result
of its activity toward PI(4,5)P2 [48] and PTPMT1 as a result of its activity toward PI(5)P [47]. The PRLs are depicted in full length; other PTPs
are shown in truncated versions according to relevance with respect to sequence alignment with the PRLs. The amino acids are colored by
polarity: A, P, V, I, W, F, L, G, M (black); S, T, Y, N, C, Q (green); D, E (red); H, K, R (blue). The consensus residue and conservation rate at
each position are shown below the sequences. An ambiguous residue is indicated as ‘X’. The putative ‘CXnE’ motif, the WPD-loop and the
active site p-loop are indicated in red squares. Protein sequences were manually associated with 3D homologous structures in STRAP
(http://3d-alignment.eu/) and the alignment was computed with CLUSTALW_3D. The alignment was manually adjusted according to the
superimposed structures.
Molecular mechanisms of the PRL phosphatases P. Rios et al.
2 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS
polybasic region adjacent to the CAAX motif, which
aids in mediating the membrane localization of PRLs
[10,31,32] and could be involved in mediating nuclear
localization [10,25], although probably not as a
bipartite nuclear localization sequence [33].
As shown in vitro and in cells for PRL-1, as well as
in vitro for PRL-3, another regulatory feature could be
the formation of trimers or other oligomers
[10,31,33,34]. Indeed, PRL-1 crystallized in trimers
[31,34]; however, the NMR structures of PRL-3
revealed a monomeric state [32,35]. This discrepancy
could be a result of the different experimental meth-
ods, although it could also mean that there are differ-
ences within the PRL family with respect to the
ability ⁄ tendency to form oligomers. PRL-1 oligomeri-
zation, which requires C-terminal farnesylation, was
reported to be necessary for its function in cells [10].
By contrast, for PRL-3, it was shown that farnesyla-
tion-dependent oligomerization decreased the in vitro
phosphatase activity toward an unnatural substrate
[33]. Owing to the more complex environment in cells,
it is likely that the measured in vitro activity of PRL-3
does not reflect the in vivo behaviour.
The structural elucidation of PRL-1 and PRL-3 also
revealed the importance of cysteine 49. This cysteine is
localized very closely to the active site cysteine in all
structures and the two cysteines can form a disulfide
bond [31,32,34,36]. As for other PTPs, this indicates
that PRLs are subject to redox-regulation, following
the same mechanism as other DSPs such as phospha-
tase and tensin homologue (PTEN) [37]. It was specu-
lated that this redox-mechanism not only regulates the
activity of the phosphatases, but also could protect the
catalytic Cys104 from further and irreversible oxida-
tion [31,32]. Skinner et al. [38] reported that the reduc-
tion potential of this disulfide bond for PRL-1 in vitro
is lower ()365 mV) than the reduction potential range
in normal cellular environments ()170 to )320 mV),
indicating that newly-synthesized PRL-1 in cells could
be oxidized and thereby inactive. Interestingly, the
same study reported that the C-terminal CAAX box
farnesylation motif (CCIQ in PRL-1) also regulates
the PRL-1 activity in vitro. When mutating the C170
and C171 residues of the CAAX motif, the resistance
to oxidation of PRL-1 was increased, mediated by con-
formational changes. Such a conformational switch
would likely also occur upon farnesylation, increasing
the catalytic activity as a result of a lower sensitivity
to oxidation. Thus, farnesylation could not only regu-
late PRL-1 subcellular localization, but also the cellu-
lar functions that are dependent on catalytic activity
[38]. Pascaru et al. [33] reported that CAAX deleted
PRL-3 also displays enhanced catalytic activity
compared to wild-type PRL-3, which suggests that the
C-terminus and farneslyation are common features for
regulating the catalytic activity of the PRL proteins.
As similar as the PRLs may appear, many intriguing
differences are already apparent. In the present review,
we first compare the structural features of the PRLs to
related phosphatases and then describe the features for
each PRL in order of how well studied they are, focus-
ing on a discussion of key characteristics and signalling
pathways, as well as the novel insights that have
appeared subsequent to previous reviews [2,3]. Next,
we discuss the differences between the PRLs. Finally, a
discussion about the catalytic site architecture of the
PRLs and its putative influence on the substrate recog-
nition mechanism is provided.
Comparison of structural featuresof the PRLs with other relatedphosphatases
In general, structural analyses revealed that the PRLs
have hydrophobic, shallow binding pockets and a wide
pocket entrance [31,32,34,35]. Compared to the struc-
turally most closely-related phosphatases [PTEN,
vaccinia H1-related phosphatase (VHR), cell division
cycle 14 homologue (CDC14), kinase-associated phos-
phatase (KAP)], PRLs lack helices and loops that can
be important for substrate recognition [31,34]. In addi-
tion, all of those phosphatases, which are structurally
most closely related, have very different substrate spec-
ificities, ranging from pTyr (VHR) [39], pSer (CDC14)
[40] and pThr (KAP) [41] to phosphatidyl inositol
phosphates (PTEN) [42], making direct conclusions
with respect to the substrate specificity of PRLs from
this general comparison impossible. For a more
detailed comparison, in Fig. 2 the crystal structure of
the catalytic pocket of PRL-1 complexed with a sulfate
ion [34] is overlaid with complexed structures of the
related phosphatases PTEN [43], VHR [44], CDC14
[45] and KAP [46], and also with PTP mitochondrial 1
(PTPMT1) [47] and the PTEN-like phosphatase
Ci-VSP from Ciona intestinalis [48,49]. The latter two
were chosen due to their ability to dephosphorylate the
5-position of phosphatidylinositol phosphates in light
of the fact that PRL-3 has been proposed to be a
phosphoinositide-5-phosphatase (see below) [50]
(unfortunately, complexed structures of PRL-3 are not
yet available). In general, the active site of PRL-1
overlays well with all of the structures. The best
matches appear to be KAP and CDC14B, whereas the
worst overlay is with VHR due to the many different
amino acids. Thus, if a conclusion can be drawn from
this comparison, PRL-1 likely prefers pThr ⁄pSer
P. Rios et al. Molecular mechanisms of the PRL phosphatases
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 3
residues similar to KAP and CDC14B. Nevertheless, it
was proposed that significant structural rearrange-
ments will likely occur upon association of PRL phos-
phatases with their physiological substrates [31], and
thus only structures of PRLs with their substrates are
able to answer the question of how these interactions
look like.
Intriguingly, PRLs do not contain the conserved
[Ser ⁄Thr] residue of the PTP active site p-loop but,
instead, an alanine is found in that position (Fig. 1). It
is thought that this alanine results in the low intrinsic
in vitro activity of the enzymes [31,32] due to the role
of this Ser ⁄Thr in the catalytic mechanism of PTPs,
aiding in the release of the phosphate from the phos-
phatase [51]. Replacement of alanine with serine
enhanced the catalytic activity towards unnatural sub-
strates such as para-nitrophenyl phosphate (pNPP) or
ortho-methylfluorescein phosphate (OMFP) [31,32,50].
This is in agreement with observations for the mouse
phosphatase LDP-2, which also carries an alanine in
the respective position [52]. However, the human
orthologue, DUSP19 (SKRP1) also displayed a low
activity toward pNPP compared to other DSPs, but
this activity was not enhanced when the alanine was
Fig. 2. Structural comparison of active site of PRL-1 bound to a sulfate ion (1XM2, white) with complexed structures of closely-related
PTPs: PTEN (1D5R, green), KAP (1FPZ, cyan), CDC14B (1OHE, yellow) and VHR (1VHR, pink). Ci-VSP (3AWF, blue) is depicted as a result
of its activity toward PI(4,5)P2 [48], and PTPMT1 (3RGQ, orange) as a result of its activity toward PI(5)P [47]. Amino acids are numbered
according to protein database files.
Molecular mechanisms of the PRL phosphatases P. Rios et al.
4 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS
replaced with a serine [53]. Curiously, an Ala to Ser
mutation in PRL-3 completely abolished phosphatase
activity toward a potential natural substrate [50]. Thus,
the role of the natural serine to alanine mutation is
not clear; it was suggested that it could be involved in
substrate recognition [52] or structural integrity [50],
although its role could also be different for every phos-
phatase that carries this mutation.
Another conserved loop in the PTP superfamily is
the WPD loop, of which the Asp acts as general acid
in the catalytic reaction [51]. In the PRL family, this
loop consists of the sequence 68 ⁄ 65WPFDD72 ⁄ 69 (where
the numbering refers to PRL-1 and PRL-3 ⁄PRL-2)
(Fig. 1) and, for PRL-1 and PRL-3, it was shown that
the catalytically active Asp is the D72 [25,32]. It is not
known whether the additional residues in this loop
have a function. However, as there are quite a few
exceptions in the DSP family with respect to the con-
servation of this loop; the conservation might actually
occur mostly in classical PTPs. For example, neither
PTEN, nor CDC14 carry a tryptophan close to the
catalytically active Asp (Fig. 1) and, in the myotubula-
rin family, the W-D motif is localized within the
p-loop [54]. Indeed, a D92A mutation in the WPD
loop in PTEN [55] and D72A in PRL-3 [50] caused
only a partial loss of activity and, for PTEN, it was
shown that the Asp does not act as the general acid in
the first step of the catalysis [55]. Interestingly, PTEN
shows a high sequence similarity in this region to the
PRLs in that the WPFDD of the PRLs aligns with
YPFED in PTEN (Fig. 1).
As noted above, PRLs carry a regulatory cysteine.
Interestingly, a natural mutation of the regulatory cys-
teine 71 in PTEN, which is mutated to tyrosine in
Cowden disease, led to a loss of phosphatase activity
toward the substrate inositol(1,3,4,5)tetraphosphate
[56]. A C49S mutation in PRL-1 led to slightly lower
activity toward pNPP [31]; by contrast, a C49A muta-
tion in PRL-3 did not lead to a change in activity
against the unnatural substrate OMFP [32]. Consider-
ing that an alanine mutation (C49A in PRL-3) intro-
duces a much less drastic change in electrostatic and
steric properties than a tyrosine mutation (C71Y in
PTEN) does, and that activities toward unnatural sub-
strates can sometimes be misleading [50], it is tempting
to speculate whether these regulatory cysteines fulfill
other tasks in the respective phosphatases, such as
maintaining structural integrity or aiding in substrate
recognition, for example through the correct position-
ing of amino acids that are involved in substrate inter-
actions or the catalytic mechanism. This idea is fueled
by another interesting consideration: PTPMT1 con-
tains the catalytically relevant ‘EEYE’ loop, in which
Glu73 and Glu76 were shown to be essential for cata-
lytic acticity and Glu76 interacts with and stabilizes
the conserved catalytic Arg in the catalytic p-loop in
the crystal structure [47]. As shown in Fig. 1, all of the
depicted regulatory cysteine-carrying DSPs have a Glu
close to this Cys, which aligns well with Glu73 and ⁄orGlu76 of the ‘EEYE’ motif either in the backbone
and ⁄or in the side chain (Fig. 3). Even in KAP, where
there is a small loop between the Cys and the Glu, the
alignment is excellent, and also Ci-VSP from a sea
squirt contains a Glu next to a Cys and aligns very
well (Fig. 3). Structurally closely-related DSPs that do
not contain a regulatory cysteine, with the exception
of PTPMT1, do not have a Glu in that position
(Fig. 1). Since mutation of the general acid of the
‘WPD loop’ in PTEN [55] and PRL-3 [50] reduced,
but did not abolish, catalytic activity toward the
(potential) natural substrate, and C71Y mutation
diminished the catalytic activity of PTEN [56], it is
worth investigating whether the acidic amino acid
adjacent to the regulatory cysteine could play an
important role in the general catalytic mechanism or
stabilize the catalytic pocket of DSPs that carry a
putative ‘CXnE’ motif (where X = any amino acid
and n = the number of amino acids between the C
and E residues; e.g. 0 for PRL-1 and PRL-3; 1 for
PTEN and CiVSP; 3 for KAP).
Fig. 3. Structural alignment of the putative ‘CXnE’ motif. The Glu
residues adjacent to the regulatory cysteines in the crystal struc-
tures of PRL-1 (green: 1XM2), PTEN (purple: 1D5R), KAP (pink,
1FPZ) and Ci-VSP (yellow: 3AWF) align well with the Glu of the
‘EEYE’ loop in PTPMT1 (cyan: 3RGQ). The structure of PTEN is in
complex with an inhibitor [L(+)-tartrate], which may be the reason
why the side chain of PTEN does not align with Glu144 of
PTPMT1, although the backbone aligns with the Glu141 of
PTPMT1. Only complexed sructures are compared here. Amino
acids are numbered according to protein database files.
P. Rios et al. Molecular mechanisms of the PRL phosphatases
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 5
PRL-3
PRL-3 expression, interacting proteins and
regulation
PRL-3 mRNA was found predominantly in the skele-
tal muscle and at moderate levels in the heart, as
shown in mouse [19] and human [57] tissues, and, in
both studies, PRL-3 was also detected in other organs
at lower levels. Interestingly, it was reported that the
expression in the heart only occurs during develop-
ment, and not in the human adult organism, as dem-
onstrated at the mRNA and protein levels [13]. This
finding could have important implications for any
potential drug discovery against PRL-3 because inhibi-
tion of PRL-3 in the adult heart could lead to cardio-
toxic effects [2,20]. Furthermore, PRL-3 was found to
be expressed in the developing blood vessels and pre-
erythrocytes [13], suggesting that PRL-3 plays an
important role in embryogenesis. In addition, Zeng
et al. [23] observed that PRL-3 is present in differenti-
ated villus epithelial cells of the small intestine in mice.
The upregulation of PRL-3 in cancer has received
the most study with respect to the three PRLs, and
was identified in colon [1,58], breast [59], gastric [60]
and ovarian [61] carcinomas. In addition, high levels
of PRL-3 appear to be associated with a poor progno-
ses and, particularly for colon cancer, high levels of
PRL-3 were shown to be predictive for the develop-
ment of liver metastatis [62]. These findings are
reviewed in detail in Bessette et al. [3]. In addition,
PRL-3 was reported to be elevated in oral and cervix
squamous cell carcinomas [63,64]. Furthermore, it was
found to be overexpressed in haematological malignan-
cies, namely in a subset of multiple myelomas [65,66]
and in acute myeloid leukaemia (AML) [67].
A few substrates have been suggested for PRL-3,
namely ezrin [68,69], elongation factor 2 (EF-2) [69],
keratin 8 [70] and integrin b1 [71], all four of which
have been reviewed [2], as well as stathmin [72] and
nucleolin [15]. Recently, we described phosphatidylino-
sitol(4,5)bisphosphate [PI(4,5)P2] as a potential natural
substrate. Although no in vivo activity against PI(4,5)P2
has yet been demonstrated, a correlation between differ-
ences of in vitro activity and phenotype in the cell
migration of wild-type PRL-3 and three PRL-3 mutants
was demonstrated. This correlation was only true
for activity against PI(4,5)P2 and not against the
unnatural substrate ortho-methylfluorescein phos-
phate [50]. Of the putative substrates, direct dephos-
phorylation was demonstrated in the case of ezrin and
PI(4,5)P2, whereas, for EF-2, keratin 8, nucleolin and
stathmin, PRL-3-dependent downregulation of the
phosphorylation level was shown in vivo, and integrin
b1 is now considered to be indirectly affected by PRL-3
[2,73]. In independent experiments, however, the influ-
ence of PRL-3 overexpression on ezrin phosphorylation
could not be confirmed, which may be a result of the
use of different cell lines [70,74]. Stathmin, nucleolin
and keratin 8 were shown to co-immunoprecipitate
with ectopic (inactive) PRL-3, but no direct interaction
with EF-2 was reported. Other direct interaction part-
ners have been identified: integrin a1 [71], cadherin
CDH22 [75] and the peptidyl prolyl cis ⁄ trans isomerase
FK506-binding protein 38 (FKBP38) [76], all discovered
in yeast two-hybrid screens, and PRL-3 itself through
potential oligomerization [10,33]. Most of the proposed
directly interacting proteins are related to the role of
PRL-3 in cell migration and invasion and are connected
in some way to the plasma membrane [ezrin, PI(4,5)P2,
integrin a1, CDH22] or to the cytoskeleton [keratin 8,
stathmin]. Noteworthy, nucleolin is localized to the
cytoplasm and nucleus and is involved in cell prolifera-
tion [15] and FKBP38 is a cytosolic protein that regu-
lates PRL-3 protein levels and proteasomal degradation
in MCF-7 and HCT116 cell lines [76].
In addition, an unbiased mass spectrometry-based
approach revealed 110 potential interacting proteins
when PRL-3 was used as a bait, 38 of which were con-
sidered to be of high confidence [77]. The identified
proteins have not yet been followed up by experimental
validation. It is striking that none of the proposed
binding partners from other studies were identified,
showing how difficult it is to validate substrates and
interacting proteins of PRL-3 (and phosphatases in
general).
PI(4,5)P2 as a substrate for PRL-3 offers a connec-
tion to another substrate, ezrin. Ezrin forms part of
the ERM (ezrin–radixin–moesin) complex, which con-
nects the plasma membrane with the actin cytoskeleton
and is implicated in tumour metastasis [78]. Ezrin
requires PI(4,5)P2 binding and Thr567 phosphorylation
to become active at the plasma membrane [79], so that
it can exert its multiple functions in cell adhesion,
motility, morphogenesis and signalling pathways
[79,80]. In addition to potential PI(4,5)P2 depletion by
PRL-3, PRL-3 is assumed to dephosphorylate Thr567
[68], meaning that PRL-3 could inactivate ezrin in
multiple ways. Considering that the binding of ezrin
by PI(4,5)P2 is required for the phosphorylation of
Thr567, the lower phosphorylation level of Thr567
could also be an indirect effect as a result of the pre-
vention of ezrin binding to the plasma membrane [79].
On the other hand, PRL-3 has been reported to upre-
gulate Src kinase activity [81] (see also below), and Src
can phosphorylate Tyr477 in ezrin, which is required
Molecular mechanisms of the PRL phosphatases P. Rios et al.
6 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS
for anchorage-independent growth and cell invasion in
a 3D environment [82]. Tyr477 phosphorylation was
crucial for the correct localization of ezrin to sub-
membraneous patches in the 3D culture. The influence
of Thr567 phosphorylation and PI(4,5)P2 binding was
not studied in this context; however, other factors
aside from the latter two are important for proper
activity and membrane localization of Ezrin, depend-
ing on the functional context. This shows that the
activity of PRL-3 takes place in a very complex envi-
ronment, which in itself remains incompletely under-
stood.
PRL-3 is subject to complex regulatory mechanisms.
It is known that PRL-3 mRNA levels do not necessar-
ily correspond to protein levels [83] and that PRL-3
abundance is controlled at the transcriptional and
translational levels, as well as through degradation
mechanisms [76] (see above).
PRL-3 is a direct transcriptional p53 target gene in
mouse (mouse embryonic fibroblast; MEF) and human
(H1299 human lung adenocarcinoma, SK-Hep-1 hepa-
tocellular carcinoma) cells [30,84], and ectopic expres-
sion of p53 and p73 increases PRL-3 transcription in
H1299 nonsmall cell lung cancer cells [85]. In other
cancer cells, such as SNU-475, Hep3B and HeLa cells,
the transcriptional level of PRL-3 did not increase
upon ectopic p53 expression [30], suggesting that this
interaction is cell type specific (although two PRL-3
introns harbour a p53 consensus sequence that can
bind the p53 protein) [84]. PRL-3 transcription is also
activated by the vascular endothelial growth factor
(VEGF) through the transcription factor myocyte
enhancer factor 2C (MEF2C) in human umbilical vein
endothelial cells (HUVEC) [86]. MEF2C binds the
promoter region of PRL-3 in vitro and in vivo, and
notably, the presence of MEF2C is critical in heart
and skeletal muscle where PRL-3 is abundant. This,
together with the distinct expression pattern in human
healthy tissues, suggests that transcription of PRL-3
could be controlled by tissue specific transcription fac-
tors [86]. However, an equal enhancement of PRL-3
protein amounts in the presence of MEF2C was not
observed. Interestingly, in PRL-3-positive nonsmall cell
lung cancer cells (NSCLC), elevated levels of VEGF
and its isoform VEGF-C were found, and high levels
of both were correlated with micro and lymphatic ves-
sel density [12], demonstrating that the expression of
PRL-3 facilitates angiogenesis [2].
Snail is a transcription factor involved in the epithe-
lial–mesenchymal transition (EMT). EMT is an impor-
tant process during development and metastasis and,
in this process, cells lose cell–cell adhesion and gain
motility. Snail is known to repress the expression of
E-cadherin, resulting in the disassembly of cell–cell
adhesion junctions and an increase of invasiveness [87].
The overexpression of PRL-3 was demonstrated to
promote EMT, and it was suggested that the action of
PRL-3 leads indirectly to the deinhibition of Snail
[75,88]. Recently, however, Zheng et al. [89] reported
that the PRL-3-encoding gene contains three potential
binding sites of Snail in the promoter region, and that
the transcriptional activity of the PRL-3 promoter was
abolished after the mutation of one Snail binding site.
Snail was suggested to regulate promoter activity and
protein expression of PRL-3 in colorectal cancer cell
lines, which appears to be contradictory to the earlier
reports. Thus, the interaction between PRL-3 and
Snail requires further investigation.
Recently, Jiang et al. [74] reported that PRL-3 is a
direct regulatory target of transforming growth factor
(TGF)b signalling in colon cancer metastasis. TGFbsignalling suppresses the metastasis of colon cancer
cells potentially by inducing stress-induced apoptosis.
It was demonstrated that TGFb signalling inhibited
the expression of PRL-3 in a mothers against decapen-
taplegic homologue (Smad) 3-dependent manner.
Because a loss of TGFb signalling occurs in 30–50%
of colon cancers, this could be a feasible mechanism
for explaining PRL-3 upregulation in colon cancer
[74].
A translational regulator of PRL-3 is polyC-RNA-
binding protein 1 (PCBP1) [83]. PCBP1 overexpression
inhibited PRL-3 expression via interaction with a
GC-rich motif at the 5¢ UTR of PRL-3 mRNA. In
clinical samples of normal and cancerous epithelia, an
inverse correlation between protein levels of PRL-3 and
PCBP1 was observed, and knockdown of endogenous
PCBP1 in HCT-116 cells inhibited tumourigenesis in
mice, indicating that PCBP1 acts as a tumour suppres-
sor in vivo [83].
Signalling pathways affected by PRL-3
The signalling pathways affected by PRL-3 have been
reviewed by Bessette et al. [3] and Al-Aidaroos and
Zeng [2]. Therefore, we only briefly describe the key
signalling effects of PRL-3 and add data that have
appeared subsequent to these reviews.
By demonstrating that PRL-3 upregulates mesenchy-
mal markers and downregulates epithelial markers, it
was shown that PRL-3 promotes EMT [75,88]. It pro-
motes EMT and cell survival by acting upstream of
phosphatidylinositol 3-kinase (PI3K) [74,89]. PI3K
signalling promotes many processes, such as cell
survival, cell proliferation or cell motility, and PI3K is
an oncogene [90,91]. PRL-3 was reported to
P. Rios et al. Molecular mechanisms of the PRL phosphatases
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 7
post-transcriptionally downregulate PTEN protein lev-
els [88]. PTEN counteracts PI3K activity by converting
phosphatidylinositol triphosphate PI(3,4,5)P3 into
PI(4,5)P2; thus, its downregulation leads to the activa-
tion of PI3K signalling. In addition, PRL-3-mediated
activation of PI3K could relieve the inhibition of the
mesenchymal marker Snail (see above) by inhibition of
glycogen synthase kinase (GSK)-3b [75,88]. Further-
more, PRL-3 was reported to promote cell survival
under growth factor deprivation stress by activating and
maintaining the activity of the PI3K ⁄Akt pathway [74].
PRL-3 was suggested to either reduce the number of
focal adhesions and ⁄or increase focal adhesion turn-
over to mediate cell invasion and motility [2]. Focal
adhesion complexes are multi-component sites where
integrins mediate the contact between the cell and the
extracellular matrix [92]. Levels of PI(4,5)P2 at the cell
membrane are crucial for regulating the dynamics of
focal adhesion complexes [92], and focal adhesion
kinase (FAK) is a key component of focal adhesion
complexes [93]. FAK integrates external signals to pro-
mote cell motility via many different pathways involv-
ing the regulation of (or interaction with) proteins
such as cadherins, Src, p130Cas, Rho-family GTPases
and ezrin [93], many of which were shown to be
affected by PRL-3. Integrin a1 and cadherin-22 were
reported to be direct interactors of PRL-3 (see above),
E-cadherin was shown to be downregulated by PRL-3
[75,88] and PRL-3 signalled via integrin b1 in LoVo
colon cancer cells leading to extracellular signal-regu-
lated kinase (ERK)1 ⁄ 2 activation [73]. Src kinase was
activated by PRL-3 via translational downregulation
of C-terminal Src kinase (Csk), which is a negative reg-
ulator of Src [81,94]. Src activation by PRL-3 led to
the phosphorylation of downstream proteins such as
signal transducer and activator of transcription
(STAT) 3 and p130CAS, and, in agreement with Peng
et al. [81], ERK1 ⁄2. In further studies, PRL-3 acti-
vated RhoC, downregulated Rac-GTP [28,88] and had
no effect on Cdc42 [28]. RhoA activity was reduced by
PRL-3 overexpression in the earlier study [28] and
enhanced in the later study [88]. These findings demon-
strate that the Rho family of GTPases act downstream
of PRL-3, and also show that the regulation is com-
plex. An interesting context in this regard is that active
ezrin recruits both positive and negative regulators of
the Rho family of GTPases [95]. Upon inactivation of
ezrin by PRL-3, these regulators could be released,
which would contribute to maintaining the active form
of the Rho GTPases and may explain the activation of
RhoA and RhoC when overexpressing PRL-3 [2].
The activity of PRL-3 against PI(4,5)P2 [50] offers
the intriguing possibility of PRL-3 regulating all of the
noted proteins upstream of FAK. This regulation,
however, is very complex and highly dynamic, with the
activation of Src and Rho GTPases on the one hand
and deactivation of ezrin and Rho GTPases on the
other. With PRL-3 being membrane bound and
PI(4,5)P2 being the highest abundant phosphoinositide
and a crucial part of the membrane in many respects
[92,96,97], this regulation essentially needs to be highly
dynamic and tightly regulated. Nevertheless, this inter-
action would destabilize focal adhesions and could
regulate focal adhesion turnover, leading to enhanced
motility and invasiveness. Further studies are necessary
to evaluate this hypothesis.
Expression and activity of matrix metalloproteinases
(MMP) is affected by PRLs. MMPs are extracellular
secreted proteins with a key function in tumour metasta-
sis [98]. Increased MMP2 (but not MMP9) activity and
expression levels have been found in PRL-3 stably trans-
fected LoVo cells [73]. PRL-3-induced invasion in these
cells was dependent on MMP2 upregulation and
ERK1 ⁄ 2 activation. PRL-3 also downregulated the
expression of the MMP2 inhibitor TIMP2, explaining,
at least in part, the activation of MMP2. Recently, Lee
et al. [99] investigated expression levels of several MMPs
in PRL-3-overexpressing colorectal DLD-1 cells.
MMP2 was also found to be enhanced, and MMP2
knockdown partially inhibited cell migration and inva-
sion. In addition, migration and invasion of DLD-1-
PRL3 cells was completely inhibited by small interfering
RNA (siRNA) knockdown of MMP7, whereas the over-
expression of MMP-7 increased migration. In agreement
with earlier studies, PRL-3 acted through oncogenic
pathways including PI3K ⁄Akt and ERK1 ⁄ 2 [99].A recent study revealed that intermediate-conduc-
tance Ca2+-activated K+ (KCNN4) channels were
upregulated in ectopically PRL-3 expressing LoVo
cells, and this upregulation was nuclear factor-jB(NF-jB)-dependent, revealing a novel pathway that
PRL-3 can interfere with. Blocking of KCNN4 chan-
nels inhibited PRL-3-induced cell proliferation and
arrested the cell cycle at the G2 ⁄M phase, indirectly
suggesting that PRL-3 facilitates G2 ⁄M transition in
this setting [100].
PRL-3 was also described to play a role in cell cycle
regulation in normal cells [84]. Tight control of PRL-3
basal expression in MEF cells appears to be important
to ensure cell cycle progression by facilitating G1 ⁄Stransition (as opposed to G2 ⁄M transition in LoVo
cancer cells). Its overexpression in MEF cells led to G1
arrest downstream of p53 via a PI3K-Akt-mediated
negative feedback loop, in which initial levels of PRL-3
activated the PI3K-Akt pathway but subsequent higher
levels of PRL-3 correlated with a decrease in activated
Molecular mechanisms of the PRL phosphatases P. Rios et al.
8 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS
Akt. A decrease of PRL-3 expression levels also led to
cell cycle arrest through increased p53 expression and
via cyclin-dependent kinase 2 (CDK2), relying on an
intact p53 pathway. Interestingly, in the global study
by Ewing et al. [77], CDK2 was found to be a PRL-3
interacting protein.
These results appear to contradict the role of PRL-3
in cancer; however, Basak et al. [84] suggested that, as
a result of multiple mutations in cancer cells, particu-
larly in later (metastatic) stages, and with p53 loss of
function being a very common mutation, high expres-
sion levels of PRL-3 might not succeed in inducing cell
cycle arrest, and other functions of PRL-3 might pre-
vail. It is now important to dissect the primary role of
PRL-3 in healthy cells, whether it is related to cell
migration, cell cycle regulation or both, and whether
the activity in cancer is a malfunction or hyperactivity
of a normal function. Min et al. [30] addressed the
ability of PRL-3 to regulate p53 in cancer cells. In
agreement with the results obtained in MEF cells,
PRL-3 upregulation in HCT116 colorectal cancer cells
led to a decrease in p53 expression; however, it did not
lead to cell cycle arrest but to inhibition of p53-medi-
ated apoptosis [30]. An earlier, more detailed study on
PRL-1 investigated the mechanism of action of PRL-1
on p53 [101], and this is discussed below. PRL-3 was
described to act on p53 through the same mechanisms
involving mouse double minute 2 (MDM2) stabiliza-
tion via PI3K ⁄Akt signalling and also increased tran-
scription of protein with a RING-H2 domain
(PIRH2), both leading to p53 inactivation [30].
PRL-3 appears to play a role in drug resistance in
AML. PRL-3 was found at elevated levels in AML
patients and, in six out of nine patient samples, the
overexpression was correlated with internal tandem
repeat duplication of fms-like tyrosine kinase 3 (FLT3-
ITD), a mutation that occurs in approximately 25% of
AML patients. Zhou et al. [67] reported that, in AML
MOLM-14-cells, PRL-3 acts downstream of FLT3-
ITD through STAT5 and STAT3 (but not through
Akt) activation and upregulation of McI-1, which is
known to contribute to a resistance to chemotherapy
when it is highly abundant. In addition, PRL-3 was
shown to bind histone deacetylase 4 in MOLM-14 cell
lysate [67].
PRL-1
PRL-1 expression, interacting proteins and
regulation
Initial studies reported that PRL-1 is expressed at high
levels in growing rat hepatic cells, rat intestinal epithe-
lia and some tumour cell lines, and also that it could
modulate cell growth or cell differentiation in a tissue-
dependent manner [17,25,102]. Expression of PRL-1
was found to be induced by the Egr-1 transcription
factor in liver regeneration and mitogen-activated cells
[103]. In normal adult human tissues, the PRL-1
mRNA expression pattern is widespread, although the
expression levels are variable in different tissues [104].
Endogenous PRL-1 was found to be expressed at
high levels in lymph node metastases of adenocarcino-
mas [105]. PRL-1 is also overexpressed in different
cancer cells (lung cancer, pancreatic cancer), conferring
increased cell motility and invasive properties that can
be counteracted when PRL-1 expression is knocked
down [14,106–108].
Although PRL-1 was first reported as a nuclear pro-
tein [17,102], it preferentially localizes (similar to the
other PRLs) in the plasma membrane and intracellular
membranes as a result of its farnesylation [10,23,25].
In mitotic cells, PRL-1 can localize to centrosomes
and the mitotic spindle in a farnesylation-independent
manner, colocalizing with a-tubulin (which physically
interacts with PRL-1 in vitro), and farnesylation defec-
tive mutants are reported to be associated with mitotic
defects [25].
Besides the interaction with a-tubulin, PRL-1 was
shown to interact with activated transcription factor
(ATF)-7 [109] (currently named ATF-5 ⁄ATF-X tran-
scription factor; a member of the ATF ⁄CREB family
of basic leucine zipper ⁄bZIP proteins). The interaction
involves the catalytic domain and a short adjacent
C-terminal region in PRL-1, as well as the bZIP
domain of ATF-7. ATF-7 was dephosphorylated
in vitro to some extent by PRL-1 and, to date, it is the
only proposed substrate for PRL-1. Recently, the
RhoA inhibitor p115 Rho-GTPase-activating protein
(RhoGAP) was described as a novel PRL-1 interacting
protein [110]. PRL-1 also interacts with different phos-
phoinositides (mainly mono- and di-phosphorylated)
and phosphatidic acid in vitro through the C-terminal
polybasic sequence, which cooperates with the farnesy-
lation to stabilize the protein at the membrane [10].
No phosphatase activity against phosphatidylinositol
phosphates was found for PRL-1 [10,111].
As noted above, another regulatory mechanism of
PRL-1 is oxidation. Endogenous PRL-1 in mammalian
retina cells and isolated retina tissue underwent revers-
ible inactivation by disulfide bond formation under
oxidative stress, and PRL-1 was reactivated by the glu-
tathione cellular redox system. Oxidative stress also
increased PRL-1 expression levels, suggesting that
PRL-1 can play additional roles in the oxidative stress
response [111].
P. Rios et al. Molecular mechanisms of the PRL phosphatases
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 9
Similar to PRL-3, PRL-1 is a p53 target. The PRL-
1-encoding gene contains a p53-binding element and
its mRNA transcription was reported to be regulated
by p53 [101].
Signalling pathways affected by PRL-1
Diverse focal adhesion components are regulated by
PRL-1. p130Cas phosphorylation and protein levels
were found to be downregulated in HeLa cells when
PRL-1 expression was knocked down or when the
PRL inhibitor thienopyridone was applied (this was
also the case for PRL-3) [112]. Another study reported
that the levels of Src and p130Cas were decreased
upon PRL-1 stable knockdown in A549 cells, whereas
no change in FAK expression was detected [106]. Nev-
ertheless, total tyrosine FAK phosphorylation and
Tyr397 phosphorylation levels were continuously ele-
vated when plating these cells in fibronectin, together
with a decrease in membrane protrusions and reduced
actin fiber extensions that could indicate decreased
adhesion turnover upon PRL-1 knockdown [106]. Fur-
thermore, ectopic overexpression of PRL-1 in HEK293
cells increased the autophosphorylation of Src and the
phosphorylation of FAK and p130Cas [108]. In addi-
tion, it was shown that the overexpression of PRL-1 in
A459 cells decreased the levels of vinculin, paxillin and
E-cadherin [14].
PRL-1 can modulate the activation of the small
GTPases RhoA, RhoC, Rac1 and Cdc42. In SW480
colon cancer cells, ectopic overexpression of PRL-1 led
to the activation of RhoA and RhoC, as well as the
inactivation of Rac1, and had no effect on Cdc42 [28].
Also, PRL-1-induced cell motility and invasion were
dependent on the effector Rho kinase (ROCK) in these
cells. As seen in SW480 cells, Nakashima and Lazo
[14] showed that the ectopic overexpression of PRL-1
in A549 lung cancer cells caused the activation of
RhoA (which depends on the PRL-1 catalytic activity)
and induced cell invasion and motility through Rho
kinase activation. PRL-1 overexpression in A549 cells
inactivated Rac1 and Cdc42, although this was inde-
pendent of PRL-1 catalytic activity, which suggested
that the PRL-1-promoted cell motility in A549 cells
was a result of RhoA activation and not dependent on
Rac1 and Cdc42 [14]. Interestingly, the stable knock-
down of PRL-1 also inactivated Rac1 and Cdc42 in
A549 cells (RhoA activation was not analyzed) but
only when the cells were plated in fibronectin [106].
These results reflect a complex and tight regulation of
the Rho proteins by PRL-1.
Recently, a mechanistic explanation about how
PRL-1 can activate ERK1 ⁄ 2 and RhoA signalling was
provided [110]. Through phage display screening, the
GTPase activating protein p115 RhoGAP was found
to be a new PRL-1 interacting partner. This interac-
tion involves a short motif within the p115 RhoGAP
SRC homology (SH) 3 domain. Interestingly, the crys-
tal structure of PRL-1 in complex with the p115 Rho-
GAP peptide identified in the screening revealed a
novel mode of interaction between the SH3 domain
and PRL-1 that is excluded from the canonical interac-
tion SH3 domain ⁄ ligand PxxP domain (which is absent
in PRL-1). This could be advantageous for further
drug development. It was observed that p115 RhoGAP
downregulates cell migration, ERK1 ⁄ 2 phosphoryla-
tion and RhoA activation in HEK293 cells, both in
PRL-1 stably transfected and in control cells. p115
RhoGAP also physically interacts with mitogen-acti-
vated protein ⁄ extracellular signal-regulated kinase
kinase kinase 1 (MEKK1) (and inhibits it) [113] and
RhoA [114]. However, when overexpressing PRL-1,
co-immunoprecipitation of p115 RhoGAP with
MEKK1 was greatly reduced, whereas ERK1 ⁄2 activa-
tion was enhanced. Furthermore, immunoprecipitated
p115 RhoGAP from PRL-1 overexpressing cells
showed lower GAP activity compared to control cells,
suggesting that PRL-1 can regulate p115RhoGAP
activity and thus RhoA activation. Furthermore, PRL-1
blocked the interaction between p115 RhoGAP and
RhoA. Thus, it appears that, through direct interaction
with p115 RhoGAP, PRL-1 plays a role in the modu-
lation of ERK1 ⁄2 and RhoA activation by sequester-
ing this negative regulator of MEKK1 and RhoA.
Whether the catalytic activity of PRL-1 has any influ-
ence in this process was not determined. Since the cat-
alytic activity is necessary for the PRL-induced cell
migration, as well as RhoA and ERK1 ⁄ 2 activation
[14,28,82], it would be interesting to investigate
whether this mode of action is based only on protein–
protein interactions.
The effects of PRL-1 on cell migration and invasion
can be partly mediated by an increased activity of
MMP2 and MMP9. Luo et al. [108] showed that
HEK293 cells stably overexpressing PRL-1 had ele-
vated levels (and activity) of MMP2 and MMP9. This
effect was mediated through the activation of Src by
increasing the phosphorylation of its Tyr416 (where
the residue number refers to chicken Src, correspond-
ing to Tyr419 in humans), leading to an increased
phosphorylation of p130Cas and FAK, and also
through the activation of ERK1 ⁄ 2. Moreover, PRL-1-
induced Src and ERK1 ⁄ 2 activation appear to control
the transcriptional upregulation of MMPs by activa-
tion of the transcription factors AP-1 and Sp-1. Simi-
lar regulation of MMPs, Src and ERK1 ⁄ 2 by PRL-1
Molecular mechanisms of the PRL phosphatases P. Rios et al.
10 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS
was found in the lung cancer cell lines A549 and
H1299, where high levels of endogenous expression of
PRL-1 correlated with increased MMP2 and MMP9
expression levels and increased Src and ERK1 ⁄ 2 acti-
vity. Decreased cell migration and invasion was
observed when the expression of PRL-1 was knocked
down or when MMP or Src activity was inhibited in
these cell lines. In addition, the ectopic overexpression
of PRL-1 induced the expression of MMP2 and
MMP9 in H1299 cells, as observed in HEK293 cells
[108].
Similar to PRL-3, PRL-1 is implicated in cell cycle
regulation. The overexpression of PRL-1 in D27 ham-
ster pancreatic ductal epithelial cells induced cell cycle
progression, promoting entry into the S phase, upregu-
lating CDK2 activity and cyclin A protein levels, and
downregulating p21Cip1 ⁄ Waf1 levels [7]. The ectopic
expression of a catalytic defective mutant in HeLa cells
showed delayed progression through mitosis but no
other effects through the cell cycle [25].
Min et al. [101] reported that PRL-1 downregulates
p53 via a negative feedback mechanism. Endogenous
and exogenous p53 levels were reduced via ubiquitina-
tion when overexpressing PRL-1 in HCT116 and HeLa
cells. In addition, p53 levels were elevated when PRL-1
expression was suppressed by siRNA. When PRL-1
was overexpressed, an increased transcription of the
p53 ubiquitin ligase PIRH2 was observed, which was
mediated by the serum response factor target EGR1
(which, in turn, was transcriptionally activated by
PRL-1 overexpression). Furthermore, an increase in
Ser473 Akt phosphorylation was observed upon PRL-
1 overexpression leading to the phosphorylation of
MDM2, which can function both as a p53 ubiquitin
ligase and an inhibitor of p53 transcriptional activa-
tion. Both PRL-1-mediated p53 degradation pathways
were found to be independent. Thus, PRL-1 and PRL-3
might contribute to tumour development by the inhibi-
tion of p53-mediated apoptosis.
PRL-2
PRL-2 expression, interacting proteins and
regulation
The human PRL-2-encoding gene was identified in the
BRCA1 locus of chromosome 1 [18]. Subsequently,
PRL-2 was also identified in mice, and northern blot
analysis of PRL-2 mRNA showed a preferential
expression in mouse skeletal muscle [19]. More
recently, by in situ hybridization, it was shown that
PRL-2 mRNA is almost ubiquitously expressed at high
levels in normal adult human tissues [104].
To date, the only reported PRL-2 interacting protein
is the b-subunit of Rab geranylgeranyltransferase II
(bGGT II) [5,24]. The geranylgeranyl transferase is a
heterodimeric enzyme composed of a and b subunits,
and incorporates C20 geranylgeranyl isoprenoids into
proteins containing a CAAX motif. The C-terminal
variable region of PRL-2 is required for the interaction
(which is specific for PRL-2 but not for PRL-1 or
PRL-3), and prenylation of PRL-2 is also necessary,
although PRL-2 is not a substrate of bGGT II [24].
This interaction was proposed to be a regulatory
mechanism of GGTII activity because the binding of
bGGT II to PRL-2 and to the aGGT II subunit is
mutually exclusive [24]. No physiological substrate has
yet been found for PRL-2.
Downregulation of the enzymatic activity by a disul-
fide bond between Cys49 and Cys104 has been demon-
strated for PRL-1 and for PRL-3. The same would be
expected for PRL-2, although this remains to be
addressed.
PRL-2 in cancer and signal transduction
Among the PRL group of proteins, PRL-2 is the least
studied member. In particular, its role in cancer has
not been addressed in depth, even though it was ini-
tially reported that the ectopic expression of PRL-2 is
involved in cell transformation and tumour progres-
sion [6]. Two other studies showed the expression of
PRL-2 in different primary and metastatic tumours
[105,115], but detailed studies about the regulation of
signalling mechanisms and PRL-2 in cancer biology
were (and are) still missing. In recent years, a number
of studies reported that PRL-2 is also overexpressed in
different cancer cell lines and ⁄or tumour samples (pan-
creatic, breast and lung cancer) and, more importantly,
it was shown that PRL-2 is associated with tumour
progression [4,5,107]. Also, an effect on malignant pro-
gression and metastasis by ectopic overexpression of
PRL-2 in hematopoietic cells was reported [116].
Taken together, these recent findings demonstrate that
PRL-2, similar to PRL-1 and PRL-3, should be con-
sidered as an oncogenic protein, and emphasize the
importance of carrying out individual studies of the
three members of this group of phosphatases.
Stephens et al. [107] found that PRL-1 and PRL-2
(but not PRL-3) are overexpressed in pancreatic cancer
cell lines and pancreatic tumours. Significant results in
decreased cell growth, cell migration and soft colony
agar formation were observed only when performing
double knockdown of PRL-1 and PRL-2 expression in
PANC1 and MIA PaCa-2 pancreatic cancer cell lines,
suggesting the overlapping functions of both proteins.
P. Rios et al. Molecular mechanisms of the PRL phosphatases
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 11
These effects can be mediated by PI3K and Erk1 ⁄ 2signalling because there was a decrease in Akt
serum-induced phosphorylation in both cell lines and
decreased Erk1 ⁄ 2 activation in MIA PaCa-2, whereas
this was increased in PANC1 cells [107].
PRL-2 mRNA levels are elevated and associated with
prognosis in pediatric AML [117]. Akiyama et al. [116]
showed that, when stably overexpressing Flag-PRL-2
into the murine pre-B cell line BaF3ER, different malig-
nant features are observed (including increased cell
migration). These cells displayed increased erytropoie-
tin and interleukin-3 dependent cell growth and, when
stimulated with erytropoietin or interleukin-3, the phos-
phorylation levels of STAT5 were two- or five-fold
higher, respectively, than the stimulated control-trans-
fected cells. Also, PRL-2 enhanced erytropoietin-
induced cell growth in mouse primary bone marrow
transduced cells. Thus, a contribution of PRL-2 in
hematopoietic malignancies was suggested, and this
may involve STAT5 mediated signalling. Because the
injection of PRL-2 overexpressing cells did not result in
tumours in nude mice, and also because PRL-2 overex-
pressing cells were still dependent on proliferation med-
iated by growth factors, PRL-2 may need additional
oncogenic factors to achieve a complete malignant phe-
notype in hematopoietic cells [116].
It was previously shown that the PRL mRNAs were
elevated in different breast cancer cell lines, although
significant differences in elevated mRNA levels
between neoplastic and normal tissue were only found
for PRL-3 [118]. Hardy et al. [5] observed that mRNA
levels of PRL-2 were elevated in primary breast
tumours compared to normal tissue levels in 16 out of
19 patients. More remarkably, PRL-2 was greatly
overexpressed in metastatic lymph nodes compared to
primary tumours. Hardy et al. [5] demonstrated that
PRL-2 plays a role in cell migration and the transfor-
mation process in different breast cancer cells. Ectopic
expression of PRL-2 in fully transformed TM15 and
DB7 cell lines increased colony formation in soft agar
and cell migration. Knockdown of PRL-2 in MDA-
MB-231 cells decreased anchorage-independent growth
and cell migration. When implanting cells overexpress-
ing PRL-2 into the mouse mammary fat pad, an
increase in tumour size and weight was observed com-
pared to control animals (which was also correlated
with increased ERK1 ⁄ 2 phosphorylation). On the
other hand, an effect in mice breast tumour generation
only took place in PRL-2 transgenic mice against an
oncogenic ErbB2 background (which exhibited acceler-
ated tumour development, increased ERK1 ⁄ 2 phos-
phorlyation and had no effect in Akt activation) [5].
Similar to the reported findings in hematopoietic can-
cer cells [116], PRL-2 alone would not be sufficient to
trigger oncogenesis.
Wang and Lazo [4] reported that PRL-2 is involved
in lung cancer cell migration and invasion through the
ERK1 ⁄ 2 signalling pathway. It was found that PRL-2
was overexpressed in four lung cancer cell lines com-
pared to the CCL202 normal fibroblast lung cell line.
When knocking down the PRL-2 expression with
siRNA, migration and invasion of A549 cells was inhib-
ited. Decreased levels of p130Cas, previously described
in Hela cells [112], and vinculin were found, whereas
paxilin levels remained unchanged upon PRL-2 knock-
down. Silenced expression of PRL-2 did not have any
effect on Src protein levels or phosphorylation status
(neither in Akt or p53) in A549 cells, but it led to a
decrease in ERK1 ⁄2 phosphorylation. In addition, ecto-
pic overexpression of PRL-2 induced cell migration, cell
invasion and Erk1 ⁄ 2 phosphorylation (and its nuclear
translocation), and both the catalytic activity and farn-
esylation were necessary. As a result of their findings
regarding Src kinase, Wang and Lazo [4] suggested that
PRL-2, compared to PRL-3 and PRL-1, signals
through different mechanisms in A549 cells (see below).
Nevertheless, Akt activation was downregulated by
PRL-2 knockdown in pancreatic cancer cell lines [107],
which reflects that the actions of PRL-2 likely depend
on the molecular context in different cancers.
Finally, similar to PRL-1, PRL-2 is involved in cell
cycle regulation by promoting the G1 to S transition
through the downregulation of p21Cip1 ⁄ Waf1 [7].
Differences in molecular mechanismsof the PRLs
Signalling pathways
Some common signalling mechanisms are shared by
the three PRLs, such as the activation of ERK1 ⁄2or the regulation of the focal adhesion contacts via
p130Cas (Fig. 4). However, some differences in focal
adhesion contact regulation by the PRLs can be
found (e.g. at the Src kinase level). Src kinase
activity is regulated by autophosphorylation at its
Tyr419 residue (meaning activation) or by phosphor-
ylation at its C-terminal Tyr530 residue (meaning
inactivation) (where residue numbering refers to the
human sequence) by Csk [119]. PRL-3 downregulates
Csk and thereby activates Src, but this mechanism has
not been studied for PRL-1 or PRL-2. However, a dif-
ferent mechanism of Src activation by PRL-1 was
observed through the increase of tyrosine phosphoryla-
tion at the Src Tyr419 residue, which was not observed
for PRL-3. Also, the protein levels of Src were
Molecular mechanisms of the PRL phosphatases P. Rios et al.
12 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS
downregulated upon PRL-1 knockdown. By contrast,
PRL-2 knockdown decreased neither the protein, nor
the phosphorylation levels of Src, although p130Cas
levels were diminished. Therefore, PRL-2 was proposed
to use a Src-independent mechanism of p130Cas signal-
ling. Further studies are needed to completely under-
stand this observation.
Differences in the regulation of Rho proteins by PRL-
1 and PRL-3 are found (the effects of PRL-2 in the Rho
GTPase family have not yet been analyzed). Both acti-
vate RhoA and RhoC; however, PRL-3 can also down-
regulate RhoA activity. PRL-3 downregulates Rac1
and, to date, an effect on Cdc42 has not been observed.
The effects of PRL-1 on Rac1 and Cdc42 are not yet
completely understood. Different modes of regulation
can be attributed to distinct cellular contexts or to the
dynamic regulation of Rho proteins during cell adhesion
and migration. Whether PRL-3 could also interact with
p115 RhoGAP (as is the case for PRL-1) and regulate
the activities of RhoA and ERK1 ⁄ 2 in this way has not
been addressed yet.
The MMPs are positively regulated by PRL-1 (via
Src ⁄ERK1 ⁄ 2 and increasing MMP2 and MMP9
expression) and by PRL-3 (via integrin b1 or
Fig. 4. Overview depicting the current knowledge of the signalling pathways affected by the PRL phosphatases and the outcome in cell
migration and proliferation. Arrows indicate positive regulation; crossed lines indicate negative regulation; and question marks indicate either
not yet understood or not studied processes. Detailed explanations are provided in the text.
P. Rios et al. Molecular mechanisms of the PRL phosphatases
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 13
PI3K ⁄Akt and ERK1 ⁄2 and increasing MMP2 and
MMP7; but not MMP9 activity and expression).
Further investigations are needed to understand which
molecular mechanisms of MMP activation are shared
by PRL-3 and PRL-1 and which are not, and no stud-
ies are available for PRL-2 in this respect.
It appears that PRL-1 and PRL-3 share a common
mechanism of p53 downregulation through the activa-
tion of the ubiquitin ligases MDM2 and PIRH2. In
addition, both PRL-1 and PRL-3 are p53 targets. PRL-
1 downregulates the cyclin dependent kinase inhibitor
p21; PRL-2 does this as well, however, there are no stud-
ies addressing the putative regulation of p53 by PRL-2.
Furthermore, the activation of the EMT has only
been studied for PRL-3 and not yet for PRL-1 or
PRL-2.
The discovery of the physiological substrates of the
PRL phosphatases and the correlation with their cellular
phenotypes is probably one of the most important ques-
tions that still remains unanswered. As noted above, the
only putative substrate identified for PRL-1 is the tran-
scription factor ATF-7 ⁄ATF-5. Subsequently, no fur-
ther studies have been carried out aiming to establish
whether ATF-7 ⁄ 5 is a bona fide substrate and to under-
stand its physiological relevance. Interestingly, both
PRL-2 and ATF-5 mRNAs were found to be overex-
pressed in the L1236 Hodgkin’s lymphoma cell line
[120]. It would be interesting to determine whether
PRL-2 is also an interacting partner of ATF-5. ATF-5 is
widely expressed in human carcinomas [121] and regu-
lates cell differentiation, cell survival and apoptosis
[122]. In glioblastoma cells, ATF-5 ⁄ 7 loss of function
lead to apoptosis [123] and the prosurvival protein
BCL-2 is a downstream target of ATF-7 [124]. Whether
phosphorylation plays a role in the regulation of ATF-
7 ⁄ATF-5, and whether it could be affected by PRL-1 (or
other PRLs), requires future studies.
Of the putative PRL-3 substrates, only phosphoinosi-
tides have been tested as substrates for PRL-1.
Strikingly, PRL-1 does not show activity against phos-
phoinositides [10,111] (V. McParland and M. Kohn,
unpublished observations), whereas PRL-3 dephosph-
orylates PI(4,5)P2 [50]. This finding could indicate,
despite sequence and structural similarities, that the
PRL phosphatases possess important differences in
function and that the presence of only a very few dissim-
ilarities in sequence and structure could make a big dif-
ference with respect to substrate specificities.
Comparison of PRL structures and sequences
What could those dissimilarities in sequence and
structure be? Figure 5 shows the NMR structures of
PRL-3 [32,35] and the X-ray crystal structures of
PRL-1 [31,34]. The different methods by which these
structures were obtained should be kept in mind
when comparing these structures because differ-
ences can occur due to the different methods
employed.
The PRL-3 structures were solved in the apo form
and, in both structures, PRL-3 is shown in the reduced
state with respect to Cys49 and Cys104. Overlaying
both PRL-3 structures shows that the WPFDD loop is
very flexible but, in both structures, the loop is not
closing the active site, suggesting that PRL-3 is in an
inactive but reduced state and potentially ready to
accept substrates (Fig. 5A). PRL-1 was crystallized in
the apo form and bound to a sulfate ion. By contrast
to PRL-3, all apo structures contain a disulfide bond
between Cys49 and Cys104, showing the enzyme in its
inactive, oxidized state (Fig. 5B). Nevertheless, the apo
structures align well with the sulfate ion bound
structures, and the flexible WPFDD loop in all cases is
in its closed form (Fig. 5C). Compared to PRL-1, and
possibly as a result of the lack of the disulfide bond,
the active site p-loop of PRL-3 adopts a different con-
formation (see Arg110) and is flatter in these structures
(Fig. 5C) [34].
X-ray structures are snapshots of proteins. However,
it is curious that all three apo structures of PRL-1
show no difference in WPFDD loop conformations or
the oxidation state. Possibly, disulfide bond formation
induces a conformational change by an unknown
mechanism, which closes the protein active site, pre-
venting substrates from binding. The preference for
PRL-1 being oxidized, whereas PRL-3 is not, might
hint at the redox potential of PRL-1 being different
from that of PRL-3.
The amino acids in the active sites of PRL-1 and
PRL-3 are completely conserved; all nonconserved res-
idues are at other sites in the proteins [34]. Differing
amino acids closest to the active site comprise Ile141
and Pro77Gly78Lys79 in PRL-3, which correspond to
Phe141 and Ser77Asn78Gln in PRL-1 (Fig. 1). It was
proposed that the difference in the three amino acids
77–79 could lead to a higher flexibility in the PRL-3
compared to the PRL-1 WPFDD loop [32,34]. In
addition, amino acids 77 and 78 in PRL-3 introduce a
higher hydrophobicity than in PRL-1 at this stretch,
and the additional proline induces conformational
restraints. Similarly, Ile141 in PRL-3 appears to be
flexible and solvent-exposed, whereas the correspond-
ing Phe141 in PRL-1 is buried in a helix in all
structures (Fig. 5C). Ile141 forms a network of
aliphatic site chains with Ile130 and Leu146, which
would not only result in a difference in the position of
Molecular mechanisms of the PRL phosphatases P. Rios et al.
14 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS
the involved helices, but also add an additional
hydrophobic interface to the surface of PRL-3. We
observed that PRL-3 prefers phosphoinositides with
long lipid chains (M. Bru and M. Kohn, unpublished
results) and, interestingly, the lipid chains are able to
reach the hydrophobic stretches (as seen from mole-
cular docking experiments, X. Li and M. Kohn,
unpublished results). Thus, in addition to the different
conformations of the p-loop and WPFDD-loop, these
hydrophobic stretches could play a role with respect to
the difference in substrate (particularly phosphoinosi-
tide) recognition by PRL-3 and PRL-1, as also previ-
ously suggested for Ile141 [32].
Conclusions
In conclusion, notwithstanding the progress made in
understanding PRL molecular mechanisms, many
questions remain unanswered. Future studies are
needed to elucidate the physiological substrates of the
PRL family. It will be important to determine whether
substrates are shared by the three PRLs or if they act
only on different substrates. Since the PRLs appear to
be very similar but show distinct differences (e.g. in
substrate specificity and expression patterns), studies
carried out under ectopic overexpression conditions
need to be considered with caution when comparing
the PRLs, and cell types need to be chosen (particu-
larly in healthy cells) according to their relevance
in vivo. Together, this will help to explain the regula-
tion and function of the PRLs not only under physio-
logical conditions, but also in the context of tumours,
and could help in the development of different thera-
peutic strategies [2].
Acknowledgements
This work was supported by the German Science
Foundation (Deutsche Forschungsgemeinschaft, DFG)
within the Emmy-Noether program for M.K., and by
the EMBL and Marie Curie Action EMBL Interdisci-
plinary Postdoc fellowships for X.L. and P.R.
Fig. 5. Structural comparison between
PRL-1 and PRL-3. (A) PRL-3 apo structures
(1V3A, white; 1R6H-model01, green). (B)
PRL-1 apo structures (1RXD, green; 1X24,
yellow; 1ZCK, magenta). (C) Overlay of
PRL-1 (1XM2, white; 1RXD, cyan; 1X24,
yellow; 1ZCK, pink) and PRL-3 (1V3A,
magenta). The sulfate ion is from 1XM2.
Amino acids are numbered according to
protein database files.
P. Rios et al. Molecular mechanisms of the PRL phosphatases
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 15
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