Kinome signaling through regulated protein–protein interactions in normal and cancer cells

Available online at www.sciencedirect.com

Kinome signaling through regulated protein–protein interactionsin normal and cancer cellsTony Pawson1,2 and Michael Kofler1

The flow of molecular information through normal and

oncogenic signaling pathways frequently depends on protein

phosphorylation, mediated by specific kinases, and the

selective binding of the resulting phosphorylation sites to

interaction domains present on downstream targets. This

physical and functional interplay of catalytic and interaction

domains can be clearly seen in cytoplasmic tyrosine kinases

such as Src, Abl, Fes, and ZAP-70. Although the kinase and

SH2 domains of these proteins possess similar intrinsic

properties of phosphorylating tyrosine residues or binding

phosphotyrosine sites, they also undergo intramolecular

interactions when linked together, in a fashion that varies from

protein to protein. These cooperative interactions can have

diverse effects on substrate recognition and kinase activity,

and provide a variety of mechanisms to link the stimulation of

catalytic activity to substrate recognition. Taken together,

these data have suggested how protein kinases, and the

signaling pathways in which they are embedded, can evolve

complex properties through the stepwise linkage of domains

within single polypeptides or multi-protein assemblies.

Addresses1 Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, 600 University

Avenue, Toronto, Ontario M5G 1X5, Canada2 Department of Molecular Genetics, University of Toronto, Toronto,

Ontario M5S 1A8, Canada

Corresponding author: Pawson, Tony ([email protected])

Current Opinion in Cell Biology 2009, 21:147–153

This review comes from a themed issue on

Cell regulation

Edited by Brian Hemmings and Nikolas Tonks

Available online 18th March 2009

0955-0674/$ – see front matter

# 2009 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.ceb.2009.02.005

IntroductionProtein kinases, by definition, exert their primary effects

through the phosphorylation of specific substrates. The

ability of protein kinases to recognize appropriate targets

depends, partly, on the sequence of amino acids that

surround the phosphorylation site, which determines

whether the substrate motif can be accommodated by

the active site [1,2]. However, such short motifs are

common in eukaryotic proteomes, and probably do not

provide sufficient information to ensure that only a given

subset of substrates are phosphorylated by a protein

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kinase within the cell. To circumvent this problem,

protein kinases have evolved additional mechanisms

for the selective recruitment of substrates, which involve

kinase–substrate interactions at surfaces that are distinct

from the active site of the kinase and the phosphorylation

motif of the substrate. For example, in the case of

cytoplasmic protein-tyrosine kinases, substrate recog-

nition typically involves non-catalytic modules such as

SH2 and SH3 domains that bind to substrates or their

associated scaffolds, and thus position the kinase domain

in the vicinity of relevant substrate sites [3–5] (Figure 1).

Non-catalytic domains may also reduce the complexity of

the potential substrate space by localizing the kinase to a

particular subcellular compartment. This is the case for

the PH domains of Tec family tyrosine kinases and the

Akt/PKB and Pdk1 serine/threonine kinases, which bind

phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), and

thus localize the kinases in which they are embedded

to the plasma membrane upon activation of the PI 30-kinase pathway [6–8]. Although these non-catalytic

domains may have originally been linked to kinase

domains to aid in susbstrate recognition, they have also

acquired an ability to both positively and negatively

regulate kinase activity; the activation of such multi-

domain kinases may therefore be coupled to their ability

to interact with their substrates. This is a convenient

device to promote kinase-substrate specificity, in the

sense that such kinases may only be fully active in the

cell when in the vicinity of their physiological substrates.

The evolution of Src tyrosine kinase regulationA classic example involves the Src tyrosine kinase, which

possesses an N-terminal membrane-localizing myristoyl

group, followed in turn by non-catalytic SH3 and SH2

domains, the kinase domain, and a short C-terminal tail

(Figures 1 and 2). Since Src is constitutively associated

with the membrane, in proximity to its substrates, it must

be held in an inactive state in the absence of an appro-

priate upstream signal. This is achieved through phos-

phorylation of a tyrosine in the C-terminal tail by the

inhibitory tyrosine kinase Csk, which promotes an intra-

molecular interaction between the SH2 domain and the

phosphorylated tail, and consequently an additional

association between the SH3 domain and the SH2-kinase

linker [9–12] (Figures 2 and 3). Although these autoinhi-

bitory interactions involving the Src SH2 and SH3

domains are distant from the kinase active site, they

nonetheless attenuate catalytic activity, partly by clamp-

ing the kinase domain in a rigid state that precludes the

dynamic motions required for substrate phosphorylation


mailto:[email protected]

http://dx.doi.org/10.1016/j.ceb.2009.02.005

148 Cell regulation

Figure 1

Domain composition of cytoplasmic tyrosine kinases. The SH2-kinase domain combination is conserved in cytoplasmic tyrosine kinases and

represents a functional unit. FABD: F-actin binding domain, F-BAR: FCH-BAR, Myr: myristoyl group, SH2: Src homology 2 domain, SH3: Src homology

3 domain.

[13]. In addition, these interactions suppress adventitious

binding of the interaction domains to other proteins. This

inhibited state can be overcome by tyrosine phosphatases

that dephosphorylate the C-terminal tail, or by external

ligands that engage the SH2 and SH3 domains, and

thereby break the inhibitory grip of the phosphorylated

Figure 2

Interdomain interactions in active and inactive conformations of cytoplasmic

(PDB: 1OPL [42��]), Csk (1K9A [41]), and Fes (3BKB [36��]), and the inactive c

All structures have been aligned at the lower lobe of the kinase domain and a

domains in red, SH3 domains in green, and linkers and C-terminal SH2 bind


tail and linker region [14,15]. In this latter case, target

recognition may be coupled to kinase activation.

Since the SH2 and SH3 domains of Src family kinases

play a dual role, through their ability to repress kinase

activity on the one hand, and to coordinately promote

substrate recognition and kinase activation on the other

tyrosine kinases. The gallery of structures includes the active form of Abl

losed form of Abl (1OPL [18]), Src (2SRC [43]), and ZAP-70 (2OZO [32��]).

re shown in identical orientation. Kinase domains are shown in blue, SH2

ing sites in gray. The aC helix of the kinase domain is depicted in yellow.


Kinome signaling through regulated protein–protein interactions in normal and cancer cells Pawson and Kofler 149

Figure 3

Activation model of cytoplasmic tyrosine kinases. Models for the closed, inactive conformations of Src, Fes, and ZAP-70 tyrosine kinases are shown

on the left, models of the opened, catalytically active states in the presence of ligands for the adaptor domains and kinase domain substrates are

shown on the right. In this active conformation, the aC helix adopts a well-defined orientation and tilts toward the kinase domain C-lobe. Kinase

domains (KD) are depicted in blue, SH2 domains in red, and SH3 domains in green. The aC helix of the kinase domain is depicted in yellow. Black filled

circles labeled with a white ‘P’ and open circles labeled with a black ‘Y’ represent phosphorylated and unphosphorylated tyrosines, respectively, of

SH2 recognition sites and substrate motifs.

(Figure 3), one might wonder which of these activities

came first in the course of evolution. This question has

been addressed by the analysis of a Src family kinase

(MbSrc1) from the unicellular choanoflagellate Monosigabrevicollis, which shares a common ancestry with multi-

cellular animals. Although MbSrc1 has a C-terminal tail

that can be phosphorylated by the M. brevicollis homolog

of Csk (MbCsk), this phosphorylation does not strongly

inhibit MbSrc1 kinase activity [16�]. This observation is

consistent with the notion that the joining of non-catalytic

SH2 and SH3 domains to tyrosine kinase domains

initially served to target kinases to their substrates.

Once covalently joined together, however, intramolecular

interactions between the non-catalytic and kinase

domains could potentially evolve to regulate their


respective binding and catalytic properties and to fine-

tune and specify the cellular functions of such enzymes

[17�]. In support of this view, the Abl cytoplasmic tyrosine

kinase has a similar arrangement of SH3, SH2, and kinase

domains to Src, and is also autoinhibited by intramole-

cular interactions of the SH2 and SH3 domains with the

kinase domain (Figure 2). However, the precise mech-

anism by which this is achieved is different for the two

kinases, since the autoinhibted conformation of Abl

involves a phosphotyrosine-independent interaction of

the SH2 domain with the back side of the large lobe of

the kinase domain, which nonetheless inhibits both the

catalytic activity of the kinase domain and phosphotyr-

osine-binding by the SH2 domain, as in the case of Src

[18]. Thus, although the SH3, SH2, and catalytic domains

of the Src and Abl kinases independently have very


150 Cell regulation

similar properties, namely binding to a polyproline type II

helix, binding to a phosphotyrosine-containing peptide

motif, and phosphorylating a tyrosine site, respectively,

they can acquire complex allosteric regulation through

mutual intramolecular interactions, and do so in ways that

are distinct for the two kinases. These differences have

implications for the selective actions of tyrosine kinase

inhibitors. For example, the specificity of the inhibitor

Gleevec/imatinib for Abl, as compared with Src, is due to

its selective binding to the autoinhibted conformation of

the Abl tyrosine kinase domain, which differs from that of

the Src kinase domain owing to their distinct modes of

inhibitory intramolecular interaction with their linked

SH2 domains [18,19].

The tethering of domains in a single polypeptide chain

has therefore emerged as a mechanism that may have

allowed the step-wise evolution of complex signaling

proteins, first by directing catalytic domains to their

appropriate targets and dictating the formation of sig-

naling pathways and networks, and second by promoting

intramolecular interactions that provide tight allosteric

control over protein function.

The control of cAMP-dependent proteinkinase by protein–protein interactionsA related mechanism to that discussed for cytoplasmic

tyrosine kinases is employed by the cyclic adenosine 30, 50

monophosphate (cAMP)-dependent protein kinase

(PKA), through its linked regulatory (R) subunits. In

the inactive state, the R subunits bind to scaffold proteins

(A kinase anchoring proteins [AKAPs]), and simul-

taneously hold the associated kinase domain in an inac-

tive conformation [20]. Upon activation of adenylyl

cyclase by signaling through a G-protein-coupled recep-

tor, a rising concentration of cAMP engages nucleotide-

binding domains on the R subunit, and thereby triggers a

massive allosteric reorganization that releases the cataly-

tic (C) subunit in an active configuration [21]. However,

the access of this active pool of C subunit to substrates is

restricted by its prior localization through the R subunit to

an AKAP scaffold, which in turn is anchored directly or

indirectly to a subset of PKA substrates [22–24]. These

analogies also extend to the termination of signaling by

Src or PKA. In the case of Src, kinase activity can be shut

off by the Csk tyrosine kinase, which is associated with a

membrane-associated scaffold protein, Pag/Cbp, through

the Csk SH2 domain [25]. This co-localization of Src and

Csk therefore modifies the kinetics of Src-mediated sig-

naling by promoting a rapid return to the inactive con-

formation. Signaling by PKA is primarily terminated by

phosphodiesterases that hydrolyze cAMP to 50-AMP,

which results in the restoration of PKA to its autoinhib-

ited state. This inhibition can be enhanced by the binding

of a phosphodiesterase to the same AKAP that associates

with PKA, as in the case of the muscle-specific AKAP

(mAKAP), which interacts both with PKA and the phos-


phodiesterase PDE4D3 [26]. This co-anchoring of PKA

and PDE4D3 enhances the downregulation of PKA in

two ways. First, the anchored phosphodiesterase will

preferentially degrade cAMP in the vicinity of the acti-

vated PKA, and second, the catalytic activity of PDE4D3

is increased upon phosphorylation by PKA, which is

promoted by the propinquity of the kinase and the

phosphodiesterase. The binding of mAKAP to both

PKA and PDE4D3 therefore creates a negative feedback

loop that results in rapid termination of PKA activity,

leading to a pulse-like stimulation of mAKAP-anchored

PKA in response to cAMP [27].

Taken together, these results indicate that the catalytic

and substrate recognition properties of protein kinases,

and the kinetics of their activation in response to

upstream signals, commonly depend on protein–protein,

protein–phospholipid, and protein–small molecule inter-

actions, often mediated by dedicated binding domains

and scaffold proteins. In the same way, following the

phosphorylation of specific substrates by tyrosine or ser-

ine/threonine kinases, the subsequent assembly of sig-

naling pathways is frequently achieved through the

binding of the resulting phosphorylation sites to the

phospho-dependent interaction domains of downstream

targets. In the following sections, we will highlight recent

progress in understanding the various mechanisms by

which interaction and catalytic domains cooperate to

regulate the activity of cytoplasmic tyrosine kinases.

Regulation of the ZAP-70 tyrosine kinase by Tcell receptor activationRecent biochemical and structural analyses of the ZAP-70

and Fes cytoplasmic tyrosine kinases have revealed the

remarkably diverse modes with which linked SH2 and

tyrosine kinase domains can interact to regulate kinase

activity and substrate recruitment. The ZAP-70 tyrosine

kinase is a crucial proximal effector of the T cell antigen

receptor (TCR), following its activation by an antigen-

MHC complex [28]. Engagement of the TCR induces

Lck, a Src family kinase, to phosphorylate motifs contain-

ing two closely spaced tyrosine sites (so-called ‘immu-

noreceptor tyrosine activation motifs’ or ITAMs), located

on the cytoplasmic tails of the non-polymorphic TCR

signaling subunits [29] (Figure 3). These bisphosphory-

lated ITAMs selectively engage ZAP-70 through its two

tandem N-terminal SH2 domains, which are followed by

a linker sequence and the kinase domain. In the active

configuration the SH2 domains are bound to a bispho-

sphorylated ITAM, and are so intimately juxtaposed that

the C-terminal SH2 domain donates several residues that

are crucial for the N-terminal SH2 domain to engage one

of the phosphotyrosine sites in the ITAM, with the

second phospho-site bound to the C-terminal SH2

domain [30]. In this state the ZAP-70 kinase domain is

free to phosphorylate docking proteins such as LAT and

SLP-76, which in turn recruit SH2-containing proteins



such as phospholipase C-g that transmit the TCR signal

[31]. However, recent structural data have shown that in

the inactive state, ZAP-70 adopts an autoinhibited con-

formation that restricts kinase activity [32��]; although the

SH2 domains are exposed for potential ligand-binding,

they are physically separated, which disrupts the phos-

photyrosine-binding pocket of the N-terminal SH2

domain. This configuration is achieved by docking of a

helical sequence located between the two SH2 domains

onto the kinase domain, with the SH2-kinase linker being

captured between this inter-SH2 sequence and the

kinase domain (Figures 2 and 3). Two regulatory tyro-

sines in the SH2-kinase linker, which become phosphory-

lated during TCR signaling, are part of the hydrophobic

environment formed by these interactions. These find-

ings have suggested a model in which binding of a

phosphorylated ITAM to the ZAP-70 SH2 domains

induces a conformational rearrangement that disrupts

the inhibitory interactions between the inter-SH2

sequence, the SH2-kinase linker and the kinase domain.

This reorganization would also expose the tyrosines in the

SH2-kinase linker for phosphorylation, probably by the

TCR-associated Lck tyrosine kinase, which would

antagonize their ability to adopt the autoinhibited con-

formation and stabilize the active state. Thus, a key

feature of ZAP-70 regulation involves the cooperative

binding of the tandem SH2 domains to a phosphorylated

ITAM motif, which stimulates kinase activity.

The SH2 and catalytic domains of the Fescytoplasmic tyrosine kinase form a functionalunitThe Fes tyrosine kinase has an N-terminal F-BAR

domain, an SH2 domain, and a tyrosine kinase domain

[33] (Figure 1). Genetic, biochemical, and functional

analysis of the oncogenic avian Fes homolog (v-Fps)

has suggested that the SH2 domain associates with the

kinase domain, and has a strongly positive effect on

catalytic activity and substrate recognition [34,35]. Struc-

tural analysis of the linked SH2 and kinase domains of the

human Fes protein has recently revealed that the SH2

domain interacts with the aC helix in the small lobe of the

kinase domain, in a fashion that promotes an active kinase

conformation [36��] (Figure 2). This active configuration

is further stabilized by the binding of a ligand to the SH2

domain; in addition, the activation loop of the Fes kinase

domain adopts an active conformation both as a result of

autophosphorylation and binding to a substrate peptide.

Such data have suggested that the Fes SH2-kinase unit is

stabilized in the active state by binding of the SH2

domain to a phosphorylated site on a polypeptide, which

then orients a substrate motif toward the active site for

phosphorylation of a second tyrosine residue (Figure 3).

Genetic and functional evidence suggests that Src family

tyrosine kinases lie upstream of Fes, and might therefore

phosphorylate sites that engage the Fes SH2 domain

[37,38]. The Fes F-BAR sequence belongs to a family


of domains that form oligomeric structures and bind to

phospholipids in the context of curved membranes

[39,40]. Interaction of the F-BAR domain with specific

membrane sites may position the Fes tyrosine kinase in

the vicinity of substrates, and indeed during FceR1

signaling in mast cells, the Fes F-BAR appears to act

in concert with the SH2 domain to promote substrate

phosphorylation and degranulation [39]. Fes therefore

represents a kinase in which the SH2 domain not only

appears to target the kinase toward its substrates but also

simultaneously stimulates kinase activity. Similar obser-

vations have been made for the SH2 and SH3 domains of

Csk, which interact with the small lobe of the kinase

domain to stimulate activity [41]. In the case of Src,

binding of the SH2/SH3 interaction domains to external

ligands relieves an autoinhibited state, and by default

releases the kinase domain in an active conformation,

with little detectable interaction between the SH2/SH3

domains and the kinase following activation. By contrast,

in Fes the ligand-bound SH2 domain is required to

promote an active configuration of the kinase domain

through intramolecular interactions.

Complex regulation of the Abl tyrosine kinaseThe Abl cytoplasmic tyrosine kinase may represent an

intermediate between Src and Fes. As noted, in its auto-

inhibited conformation the Abl SH2 and SH3 domains

interact with the kinase domain and SH2-kinase linker to

inhibit kinase activity. Upon activation, however, the

SH2 domain appears to adopt a new interaction with

the kinase domain, involving the small, rather than the

large lobe [42��] (Figure 2). In cells, this novel Abl SH2–kinase interaction appears important for full kinase acti-

vation [36��,42��], although the effect is less pronounced

than for Fes and the mechanistic basis remains unclear.

This apparent coupling of the Abl SH2 and kinase

domains in the active state may be important for the

ability of Abl to mediate processive multi-site phos-

phorylation of substrates such as p130cas.

ConclusionTaken together, these data are consistent with a general

scheme in which the activation of cytoplasmic tyrosine

kinases is coupled to substrate recognition. They

suggest that intramolecular interactions between linked

interaction and catalytic domains have evolved to con-

trol the precise circumstances under which a cyto-

plasmic tyrosine kinase becomes active, and show

that although the domains of the various kinases retain

very similar intrinsic functions in binding or phosphor-

ylating exogenous proteins, they have evolved a wide

range of intramolecular regulatory interactions (Figures

2 and 3). Related mechanisms are used to direct serine/

threonine protein kinases such as PKA to their sub-

strates, and to control the kinetics of kinase activation.

These observations have interesting implications for

understanding the mechanisms of action of existing


152 Cell regulation

kinase inhibitors, and potentially for the design of novel

types of inhibitors.

AcknowledgementsWork in the authors laboratory is supported by the Canadian Institutes forHealth Research (CIHR) (grants #MOP-13466, #MOP-6849, and #MOP-57793), the National Cancer Institute of Canada and Genome Canadathrough Ontario Genomics Institute. T Pawson is a CIHR distinguishedinvestigator.

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This paper not only shows how the SH3-SH2 unit of the Abl kinase bindsto the back side of the kinase domain to inhibit catalytic activity but alsoidentifies a conformation that probably corresponds to an active state inwhich the domains are in an extended configuration, and in which the SH2domain interacts with the kinase domain.

43. Xu W, Doshi A, Lei M, Eck MJ, Harrison SC: Crystal structures ofc-Src reveal features of its autoinhibitory mechanism. Mol Cell1999, 5:629-638.


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