TRPM7 Regulates Myosin IIA Filament Stability and Protein Localization by Heavy Chain Phosphorylation

doi:10.1016/j.jmb.2008.02.057 J. Mol. Biol. (2008) 378, 790–803

Available online at www.sciencedirect.com

TRPM7 Regulates Myosin IIA Filament Stability andProtein Localization by Heavy Chain Phosphorylation

Kristopher Clark1, Jeroen Middelbeek1,2, Edwin Lasonder3,Natalya G. Dulyaninova4, Nick A. Morrice5, Alexey G. Ryazanov6,Anne R. Bresnick4, Carl G. Figdor1 and Frank N. van Leeuwen1,2⁎

*Corresponding author. LaboratoryUniversity Nijmegen Medical [email protected] used: MHCII, myo

MALDI–TOF, matrix-assisted laser dquadrupole–Fourier transform; LC-Mfluorescent protein; F-actin, filamengreen fluorescent protein; PBS, phos

0022-2836/$ - see front matter © 2008 E

Deregulation of myosin II-based contractility contributes to the pathogen-esis of human diseases, such as cancer, which underscores the necessity fortight spatial and temporal control of myosin II activity. Recently, wedemonstrated that activation of the mammalian α-kinase TRPM7 inhibitsmyosin II-based contractility in a Ca2+- and kinase-dependent manner.However, the molecular mechanism is poorly defined. Here, we demon-strate that TRPM7 phosphorylates the COOH-termini of both mouse andhuman myosin IIA heavy chains—the COOH-terminus being a region thatis critical for filament stability. Phosphorylated residues were mapped toThr1800, Ser1803 and Ser1808. Mutation of these residues to alanine andthat to aspartic acid lead to an increase and a decrease, respectively, inmyosin IIA incorporation into the actomyosin cytoskeleton and accordinglyaffect subcellular localization. In conclusion, our data demonstrate thatTRPM7 regulates myosin IIA filament stability and localization byphosphorylating a short stretch of amino acids within the α-helical tail ofthe myosin IIA heavy chain.

© 2008 Elsevier Ltd. All rights reserved.

1Department of TumorImmunology, Nijmegen Centrefor Molecular Life Sciences,Radboud University NijmegenMedical Centre, P.O. Box 9101,6500 HB Nijmegen,The Netherlands2Laboratory of PediatricOncology, Nijmegen Centre forMolecular Life Sciences,Radboud University NijmegenMedical Centre, P.O. Box 9101,6500 HB Nijmegen,The Netherlands3Centre for Molecular andBiomolecular Informatics,Nijmegen Centre for MolecularLife Sciences, RadboudUniversity Nijmegen MedicalCentre, P.O. Box 9101, 6500 HBNijmegen, The Netherlands4Department of Biochemistry,Albert Einstein College ofMedicine, 1300 Morris ParkAvenue, Bronx, NY 10461,USA5MRC Protein PhosphorylationUnit, James Black Centre,University of Dundee, DowStreet, Dundee DD1 5EH,Scotland, UK

of Pediatric Oncology, Nijmegen Centre for Molecular Life Sciences, Radboude, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail address:

sin II heavy chain; GST, glutathione S-transferase; WT, wild type; KD, kinase dead;esorption/ionization time of flight; MS, mass spectrometry; LTQ-FT, linear ion trapS/MS, nano-liquid chromatography tandem mass spectrometry; YFP, yellow

tous actin; PKC, protein kinase C; CKII, casein kinase II; HA, hemagglutinin; GFP,phate buffered saline.

lsevier Ltd. All rights reserved.

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791TRPM7 Regulates Myosin IIA Assembly

6Department of Pharmacology,University of Medicine andDentistry of New Jersey–RobertWood Johnson Medical School,675 Hoes Lane, Piscataway, NJ08854, USA

Received 27 September 2007;received in revised form15 February 2008;accepted 25 February 2008Available online4 March 2008

Edited by J. Karn
Keywords: actin; myosin IIA; cytoskeleton; phosphorylation; TRPM7
Introduction

The actomyosin cytoskeleton plays a vital role inmaintaining the structural integrity of cells and inproducing the force necessary to accomplish basiccellular functions, such as cytokinesis and migra-tion.1–3 Myosins are a diverse superfamily of actin-based motor proteins that convert chemical energyinto mechanical force.4 In muscle and nonmusclecells,myosin II is themajormotor protein driving cellcontraction. Notably, the level of myosin II-basedcytoskeletal tension generated inside the cell must becarefully controlled during cell behavior.5–7 Dereg-ulation of pathways controlling myosin II activitycontributes to the pathogenesis of several humandiseases, including cancer.8

Myosin II is a hexameric protein composed of twoheavy chains, two essential light chains and tworegulatory light chains.9 To accomplish its cellularfunction, myosin II assembles into bipolar filamentsthrough electrostatic interactions between the α-helical rod domain of its heavy chains.10 The motordomains on each end of a bipolar filament pull to-gether oppositely oriented actin filaments to gen-erate cortical tension. Three nonmuscle myosin IIisoforms have been identified in mammalian cells;these are termed nonmuscle myosins IIA, IIB andIIC. These three isoforms have overlapping functionssince defects due to the knockdown of a singlenonmuscle myosin II isoform can be rescued byoverexpressing the other isoforms.11 However, accu-mulating evidence suggests that the differentmyosinII isoforms also have nonredundant roles. Severalstudies have identified differences in mRNA expres-sion, protein localization, enzymatic properties ofthe motor domain and binding partners between thethree myosin II isoforms.12–20 Accordingly, mouseknockouts for nonmuscle myosins IIA and IIB showdistinct phenotypes.21,22

In mammalian cells, phosphorylation of the regu-latory light chains is the predominant mechanismregulating myosin II activity.3,23 Phosphorylation ofthe regulatory light chains stabilizes myosin II fila-ments, strengthens the actin–myosin association andactivates the ATPase activity of the motor domain,

leading to an increase in actomyosin contractility.However, strong experimental evidence exists foradditional regulatory mechanisms, including myo-sin II heavy chain (MHCII) phosphorylation.24–27Recently, we identified a novel signaling pathway

downstream of the bradykinin receptor that regulatesmyosin II-based contractility in nonmuscle cells.28,29Bradykinin stimulation of neuroblastoma cells acti-vates TRPM7, promoting its association with myosinIIA in a Ca2+- and kinase-dependent manner, whichleads to actomyosin relaxation and remodeling of celladhesion structures. Since TRPM7 is related toDictyostelium α-kinases,30 which control the assemblyand localization of myosin II by phosphorylating itsheavy chain,31,32 we proposed that TRPM7may use asimilar mechanism to inhibit myosin II-based con-tractility in mammalian cells. Indeed, we found thatTRPM7 can phosphorylate associated myosin IIA.28

However, it is unclear if and how myosin IIA phos-phorylation contributes to TRPM7-mediated cytos-keletal relaxation. Therefore, we investigated whe-ther TRPM7 may affect actomyosin relaxation bydirectly phosphorylating MHCIIA to regulate fila-ment stability.

Results

TRPM7 phosphorylates the region required forassembly of myosin IIA filaments

Previously, we demonstrated that TRPM7 phos-phorylates the mouse MHCIIA.28 To assess the con-sequences of this phosphorylation event on myosinIIA function, we set out to map the residues phos-phorylated by TRPM7 within the MHCIIA. Theassembly of nonmuscle myosin II isoforms requiresthe extreme COOH-terminus, which is the only po-sitively charged region.33,34 Therefore, this region ispredicted to be highly affected by phosphorylation, ahypothesis supported by the fact that other regu-latory proteins, including kinases, target this re-gion.20,25 Therefore, we investigated whether TRPM7could phosphorylate the extreme COOH-terminus ofthe coiled-coil domain of the MHCIIA. To this end,

792 TRPM7 Regulates Myosin IIA Assembly

different regions of the mouse MHCIIA were ex-pressed as glutathione S-transferase (GST) fusionproteins (Fig. 1) and tested for their ability to bephosphorylated by TRPM7 in in vitro kinase assays(Fig. 2). Wild-type (WT) but not kinase-dead (KD)TRPM7 phosphorylated the COOH-terminus(amino acids 1795–1960) and different fragmentsderived from this region of mouse myosin IIA,including both helical and nonhelical components.In contrast, TRPM7 did not phosphorylate otherfragments that were derived from areas locatedfurther towards the N-terminus of the MHCIIA.These results demonstrate that TRPM7 phosphor-ylates the positively charged COOH-terminus of theMHCIIA.

TRPM7 phosphorylates mouse and humanmyosin IIA

Certain kinases, such as calmodulin-dependentprotein kinase II, specifically phosphorylate themouse MHCIIA but not the human MHCIIA.35 Thedifferences appear to arise due to a lack of sequenceconservation within the nonhelical tail. We thereforedetermined whether TRPM7 could also phosphor-ylate the COOH-terminus of humanMHCIIA (Fig. 3).In vitro kinase assays showed that TRPM7 efficientlyphosphorylates the COOH-termini of both mouseand human MHCIIA. Phosphorylation of MHCIIAwas detectable within 1 min and increased progres-sively until it reached a maximum at about 10 min(Fig. 4). One notable difference was observed bet-ween mouse and human myosin IIA: While TRPM7could phosphorylate the nonhelical tail of mouseMHCIIA, TRPM7 did not phosphorylate this regionwithin the human homologue. Thus, our results showthat TRPM7, in contrast to calmodulin-dependentprotein kinase II, phosphorylates the coiled-coil do-main of both mouse and human MHCIIA.

TRPM7 phosphorylates Thr1800, Ser1803 andSer1808 in human myosin IIA

A multidisciplinary approach involving bioinfor-matics and proteomics was used to map the phos-

phorylation sites in the humanMHCIIA. Recently, analgorithm was developed to predict residues phos-phorylated by TRPM7 in its substrates (details of thealgorithm will be published elsewhere; A. Ryazanovet al., unpublished results). This algorithm calculatedthat Ser1803 was the residue with the highest pro-bability to be phosphorylated by TRPM7. The phos-phorylation sites in the MHCIIAwere mapped usingtwo distinct approaches to further substantiate thesepredictions. Initially, 32P-labeled tryptic peptidesfrom TRPM7-phosphorylated human MHCIIA wereseparated by reversed-phase HPLC. These peptideseluted off the column into three fractions at 33%, 43%and 54% v/v acetonitrile (data not shown). Whenpeptides from themajor peak (54%) were subjected tosolid-phase sequencing, a burst of 32P release wasobserved after the 12th cycle of Edman degradation(Fig. 5a). The molecular mass of the peptide (1822.79)as determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF)mass spectro-metry (MS) was identical with that expected for thetryptic phosphopeptide comprising the last threeresidues of GST fused to residues 1795–1806 ofMHCIIA and phosphorylated at Ser1803. All theother fractions also contained peptides phosphory-lated at Ser1803. However, mutation of Ser1803 toalanine reduced the incorporation of phosphate byonly 70% compared with WT, suggesting the pre-sence of additional phosphorylation sites within theMHCIIA (Fig. 6). We therefore further analyzed tryp-tic and GluC digests of phosphorylated MHCIIA byhigh-mass-accuracy MS using a linear ion trap qua-drupole–Fourier transform (LTQ-FT) mass spectro-meter. Several monophosphorylated peptides weredetected using the LTQ-FT in nano-liquid chromato-graphy tandem MS (LC-MS/MS) by the loss ofH3PO4 duringMS/MS, andMS/MS spectral analysisof these peptides determined that, in addition toSer1803, Thr1800 and Ser1808 are also phosphory-lated by TRPM7 (Fig. 5b and c). Mutational analysisdemonstrated that all three residues contribute to theoverall phosphorylation of myosin IIA, with Ser1803being the major phosphorylation site (Fig. 6a and b).The reduction in phosphorylation is not due tochanges in MHCIIA structure since mutation of an

Fig. 1. Fragments of theMHCIIAthat served as substrate in TRPM7kinase reactions. A schematic dia-gram of the MHCIIA is depicted,with the coiled-coil domain span-ning amino acids 1091 to 1924. TheMHCIIA ends with a short nonheli-cal tail piece (amino acids 1924–1960). For each myosin IIA frag-ment, the first and last amino acidsare indicated on either side of theline. Above the line is the total num-ber of threonine and serine residueswithin each fragment.

Fig. 2. TRPM7 phosphorylatesthe COOH-terminus of mouseMHCIIA. Different regions of thecoiled-coil domain of mouseMHCIIA were expressed as GSTfusion proteins. The purifiedrecombinant proteins were incu-bated with WT or KD TRPM7 inthe presence of [γ-32P]ATP. Theproducts of the kinase reactionwere separated by SDS-PAGE, andthe gel was stained with Coomassiebrilliant blue (bottom panel). Phos-phorylated proteins were detectedby autoradiography (top panel).


irrelevant residue (Thr1810) had no effect on the effi-ciency of MHCIIA phosphorylation.To address the differences in phosphorylation bet-

ween mouse and humanMHCIIA, we also analyzedmouse MHCIIA using LC-MS/MS. We detectedseveral phosphopeptides and mapped the phospho-rylation sites to Ser1799, Ser1803 and Thr1931. No-tably, Thr1931 is present in the nonhelical tail ofmouse MHCIIA but not human MHCIIA (Fig. 7),which explains the differences observed in in vitro

Fig. 3. TRPM7 phosphorylates the α-helical tail but notthe nonhelical tail of human MHCIIA. The COOH-terminiand nonhelical tails of mouse and human MHCIIA werepurified as GST fusion proteins and incubated with WTorKD TRPM7 in the presence of [γ-32P]ATP. The proteinswere separated by SDS-PAGE and visualized by stainingthe gel with Coomassie brilliant blue (bottom panel). Phos-phorylated proteins were detected by autoradiography(top panel).

kinase assays (Fig. 3). Mutations of Ser1803 andThr1931 led to a significant reduction in 32P incor-poration, demonstrating that these two residues arethe major phosphorylation sites in mouse MHCIIA(Fig. 6c and d). Alignment of the COOH-termini ofMHCIIA, MHCIIB and MHCIIC shows that Ser1803is conserved in myosin IIB, whereas Ser1808 is con-served in all three myosin II isoforms (Fig. 7). Weconclude that TRPM7 phosphorylates a conservedstretch of amino acids in the coiled-coil domain ofnonmuscle myosin II isoforms.

Mutation of phosphorylation sites affectsfilament stability and protein localization

To investigate the consequences of phosphoryla-tion on myosin IIA filament assembly, we generatedmyosin IIA rod constructs with the phosphorylationsites (Thr1800, Ser1803 and Ser1808) mutated to ala-nine or aspartic acid. TheWTandmutantmyosin IIArods were assembled into filaments at different NaClconcentrations, and the degree of solubility wasmeasured. Myosin IIA rods containing phosphomi-metic aspartic acid mutations showed increasedsolubility at physiological salt concentrations incomparison with WT myosin IIA and the alaninemutant (Fig. 8a). At 150 mM NaCl, 50% of the 3×Dmutant remains in the supernatant, whereas only25% of the WT myosin IIA as well as 3×A mutant issoluble. The differences in myosin IIA assemblybetweenWTand 3×D rods are further demonstratedby the midpoint values (Fig. 8b).The same mutations were introduced into a

yellow fluorescent protein (YFP)–MHCIIA constructto study the effect of these phosphorylation sites onmyosin IIA stability in vivo. In addition, the last170 aa of the COOH-terminus in YFP–MHCIIA(ΔC170) were deleted to serve as a positive controlfor myosin IIA filament disassembly.33 Subse-quently, these constructs were expressed in COS7cells because they predominantly express MHCIIB

Fig. 4. Kinetics of myosin IIAphosphorylation by TRPM7. PurifiedTRPM7 was mixed with 2 μg of GST–myosin IIA, and the reaction wasinitiated by the addition of 0.1 mMATP containing 5 μCi of [γ32P]ATP.The reaction proceeded at 30 °C forthe indicated times, after which20 mM EDTA was added to stopthe kinase reaction. Proteins wereresolved on 10% SDS-PAGE gel anddetected by Coomassie staining(bottom panel). PhosphorylatedTRPM7 and GST–myosin IIA wererevealed by autoradiography (topand middle panels).


and no MHCIIA. Western blotting showed that allconstructs were expressed to similar levels in COS7cells. However, biochemical fractionation of the cellsrevealed significant differences in the degree offilament assembly between the various MHCIIAmutants (Fig. 9). The majority of WT YFP–MHCIIAassembles into the actomyosin cytoskeleton,whereas deletion of the last 170 aa prevents thisassembly. Notably, mutations of the phosphoryla-tion sites to alanine (3×A) and aspartate (3×D) led toa decrease and an increase in YFP–MHCIIA solubi-lity, respectively (Fig. 9b). These findings identifyThr1800, Ser1803 and Ser1808 as novel regulatorysites of myosin IIA filament assembly.Based on the results of our biochemical experiments,

we hypothesized that the various YFP–MHCIIA pro-teins have different distribution patterns in COS7 cells.Indeed, we observed that WT YFP–MHCIIA co-localized with the cortical filamentous actin (F-actin)cytoskeleton, whereas the ΔC170 mutant was homo-genously distributed throughout the cytosol (Fig. 10a).Although the 3×A YFP–MHCIIA mutant also loca-lized with the cortical F-actin cytoskeleton, the 3×DMHCIIA protein formed disorganized fibrillar assem-blies within the cell body that failed to incorporate intocytoskeletal structures. In order to quantify differencesobserved between these myosin IIA tail mutants, wemonitored the extent of cortical localization (corticalindex), as was described earlier to determine sub-cellular localization of myosin IIB.26 Measurement ofthe cortical index confirmed the distribution of thevarious YFP–MHCIIA recombinant proteins, withboth 3×D and ΔC170 mutants showing reduced cor-tical localization in comparison with WT myosin IIAand the 3×A mutant. These differences most probablyreflect a differential ability of these mutants to in-corporate into functional actomyosin assemblies (Fig.10b). It should be noted that the distribution patternsof WT and mutant YFP–MHCIIA are consistent withfilament stability observed in vitro. Notably, the abilityof the 3×D MHCIIA mutant to self-assemble intodisorganized fibrillar structures is represented by itsrelative insolubility in Triton X-100 in comparisonwith the deletion mutant ΔC170, but its inability toassociate with cytoskeletal structures leads to an in-

crease in solubility in comparison with WT MHCIIA.Thus, the different assays to monitor assembly ofmyosin IIA filaments both in vitro and in vivo are ingood agreement. Phosphomimetic mutations lead toproteins that require lower NaCl concentrations todisrupt filaments in vitro, are more soluble in Triton X-100 and show lower cortical indices compared withWT MHCIIA in vivo. In contrast, mutations that pre-vent phosphorylation have the opposite effect. Wetherefore conclude that phosphorylation of Thr1800,Ser1803 and Ser1808 leads to a redistribution of theMHCIIA filaments in cells away from the cortexand prevents their incorporation into cytoskeletalstructures.

Discussion

In this study, we addressed themolecular mechan-ism by which TRPM7 directly affects the activity ofnonmusclemyosin IIA.We demonstrate that TRPM7phosphorylates Thr1800, Ser1803 and Ser1808 in thecoiled-coil domain of the MHCIIA and that phos-phomimetic mutations introduced at these sites leadto the disassembly of myosin IIA filaments whenexpressed in cells. To the best of our knowledge, thisstudy and another recent investigation36 are the firstto show that phosphorylation of the MHCIIA affectsfilament assembly in vivo. Moreover, our resultsindicate a conservation of function between Dictyos-telium and mammalian α-kinases.We have chosen to investigate the effect of TRPM7

on the nonmuscle myosin IIA isoform rather thanthe myosin IIB or IIC isoforms for several reasons.First, both TRPM7 and myosin IIA are ubiquitouslyexpressed, whereas myosins IIB and IIC have amorerestricted expression pattern.12,37,38 For instance,both TRPM7 and myosin IIA are widely expressedin hematopoietic cells, whereas myosins IIB and IICare absent. Second, previous work had shown thatTRPM7 associates with the nonmuscle myosin IIAisoform in a Ca2+- and kinase-dependent manner,leading to actomyosin relaxation.28 Third, nonmus-cle myosin IIA regulates cell adhesion in neuronalcells. Knockdown of myosin IIA but not myosin IIB

Fig. 5. Mapping of phosphorylation sites inMHCIIA. (a) Identification of Ser1803 by Edmandegradation sequencing of32P-labeled peptides. GST–MHCIIA COOH-terminus was incubated with TRPM7 in the presence of [γ-32P]ATP andsubjected to SDS-PAGE. The phosphorylated GST–MHCIIA fusion protein was excised from the gel and digested withtrypsin, and the peptide mixture was separated by HPLC on a C18 column. The phosphopeptides were sequenced byEdman sequencing, with 32P radioactivity being measured after each cycle of degradation. The phosphorylated residuewas assigned by a combination of solid-phase Edman sequencing and MALDI–TOF. (b) Identification of phosphorylationsites by LC-MS/MS. Phosphorylated GST–MHCIIA COOH-terminus was digested with trypsin, and peptides wereseparated on a nano-LC C18 column, which was connected inline with a high-mass-accuracy LTQ-FT mass spectrometer.RepresentativeMS2 andMS3 spectra of a peptide phosphorylated at position Ser1808 are depicted (m/z observed of parention=637.8227, mass accuracy=0.82 ppm, +2 charge state; NL indicates neutral loss of H3PO4, which triggers acquisition ofMS3 spectrum). (c) Summary table of the phosphorylation site results by LC-MS/MS.


leads to the disruption of focal adhesions formed inneuronal cells.39 Recently, we have shown that acti-vation of TRPM7 leads to remodeling of adhesionstructures in neuronal cells and that this phenotypecan be mimicked by pharmacological inhibition ofmyosin II.28 Taken together, these results suggestthat TRPM7 may be influencing myosin IIA func-tion. However, we did observe that several phos-phorylation sites (Ser1803, Ser1808) are conserved inmyosins IIB and IIC. Therefore, we are currentlyaddressing the potential role of TRPM7 in regulatingmyosins IIB and IIC.

TRPM7 associates with the actomyosin cytoskele-ton where it phosphorylates theMHCIIA but not theassociated myosin light chain (Supplementary Fig.S1). Here, we extend on these findings by demon-strating that TRPM7 phosphorylates the extremeCOOH-terminus of the MHCIIA. Interestingly, thisregion of the MHCIIA is critical for filament as-sembly,33 and this can be regarded as an importantregulatory domain. Net charge analysis of theMHCIIA shows that the region phosphorylated byTRPM7 lies within the only positively charged areaof the coiled-coil domain (data not shown). This

Fig. 6. Ser1803 is the major phosphorylation site in the COOH-termini of the coiled-coil domains of both human andmouse myosin IIA. (a) Mutations of Thr1800, Ser1803 and Ser1808 in human MHCIIA reduce phosphorylation to back-ground levels. The phosphorylated residues mapped in GST–human MHCIIA COOH were mutated to alanine indivi-dually or in combination. Since substrate recognition by α-kinases may be influenced by the secondary structure, Thr1810,which was not identified by MS or predicted to be phosphorylated, was also mutated as a negative control. GST–humanMHCIIA proteins were incubated with TRPM7 in the presence of [γ-32P]ATP and subjected to SDS-PAGE. Equal loadingof the GST fusion proteins was verified by Coomassie staining (top panel), and phosphorylated proteins were detected byautoradiography. The phosphorylation of the different GST–human MHCIIA proteins by TRPM7 is depicted in themiddle panel. The presence of equal levels of kinase activity in each sample was determined by monitoring TRPM7autophosphorylation (bottom panel). (b) Quantification of the degree of phosphorylation of the different GST–humanMHCIIA fusion proteins by TRPM7 using phosphorimager analysis. The level of 32P incorporation into the WT GST–humanMHCIIA fusion protein was set to 1, and phosphorylation of all other proteins is reported relative to this value. (c)Ser1803 is the major phosphorylation site in the helical region of mouse myosin IIA. GST–mouse MHCIIAwas mutated atpositions Ser1803 and Thr1931 either individually or in combination. Phosphorylation of these GST–mouse MHCIIAmutants by TRPM7 was compared with WT GST–mouse MHCIIA by in vitro kinase assay as described in (b). (d)Phosphorylation of different mouse MHCIIA proteins was quantified as described in (b).


region was recently found to be critical to myosin IIBfilament assembly,34 and, based on its charge, wepredict that it will be highly responsive to phospho-rylation. Accordingly, a number of protein kinases,including protein kinaseC (PKC) and casein kinase II(CKII), have been found to phosphorylate this regionof the helical tail, leading to a decrease in the criticalsalt concentration required to solubilize myosin IIAfilaments in vitro.25 Moreover, point mutations thatalter the charge composition of the MHCIIA tailaffect myosin IIA filament assembly, leading to

human disease.40 Finally, other regulatory proteins,such as S100A4, also target this area of theMHCIIA.20 Thus, TRPM7 specifically phosphory-lates the region important for the regulation ofmyosin IIA filament formation.Our experiments identify Thr1800, Ser1803 and

Ser1808 as novel residues important for regulatingmyosin IIA assembly. These three TRPM7 phosphor-ylation sites are concentrated in a short stretch ofamino acids, rather than being scattered throughoutthe COOH-terminus of the MHCIIA, demonstrating

Fig. 7. Conservation of phosphorylation sites betweenmouse, human andDictyosteliummyosin II. (a) Alignment of themouse MHCIIA and human MHCIIA, MHCIIB and MHCIIC COOH-termini. The phosphorylation sites are underlined,and conserved amino acids are shown in boldface. Note that two of three residues in the conserved stretch of amino acidswithin the coiled-coil domain of humanMHCIIAwere verified inmouseMHCIIA by LC-MS/MS. Alignment with humanMHCIIB andMHCIIC reveals that themajor phosphorylated residue in humanMHCIIA, Ser1803, is conserved inMHCIIBbut not in MHCIIC, whereas Ser1808 in mouse and human MHCIIA is conserved in both human MHCIIB and MHCIIC.


that the kinase reactions are highly specific. More-over, the conservation of these residues acrossspecies and among nonmuscle myosin II isoformsprovides further evidence that they represent im-portant regulatory sites. Based on this conservation,we propose that Ser1803 and Ser1808 are expected tobe the most important regulatory sites. Accordingly,we found that mutating Ser1803 was already suffi-cient to affect the subcellular localization and TritonX-100 solubility of MHCIIA (Supplementary Figs. S2and S3).

Fig. 8. Mutation of phosphorylation sites to aspartic acid affemutant rods wasmonitored using a standard sedimentation assayfit to the Hill equation. (b) Midpoint measurement for WT and mfor two to three independent experiments.

Although the differences in solubility betweenWTand mutant myosin IIA are relatively modest, theyare highly reproducible. Importantly, mutation toalanine and that to aspartic acid produce oppositeeffects on myosin IIA filament stability, whichstrengthens the notion that these residues regulatefilament-forming properties. A possible explanationfor the relatively small differences between the va-rious recombinant myosin IIA proteins is that themutations, particularly, mutations to aspartic acid(3×D),may not fullymimic the phosphorylated state.

cts myosin IIA filament stability. (a) The assembly of WTand. (•) WT; (○) 3×A; (▪) 3×D. The solid lines represent the bestutant myosin IIA rods. Data are reported as the mean±SEM

Fig. 9. Mutation of phosphorylation sites to aspartateincreases Triton solubility of myosin IIA. YFP–MHCIIAconstructs were transiently transfected into COS7 cells. Thecells were fractionated into Triton-soluble and -insolublefractions. Equal amounts of these fractions were separatedby SDS-PAGE and immunoblotted. Exogenous myosin IIAwas detected using anti-GFP antibodies, followed by IRDye800-coupled antimouse immunoglobulin G secondaryantibodies. Fluorescence was measured using an OdysseyInfrared Imaging System. (a) Representative Western blotdepicting the presence of YFP–MHCIIA mutants in thedifferent fractions. (b) Quantification of the degree of solu-bility of each YFP–MHCIIA fusion protein. The data arepresented as the percentage of soluble MHCIIA protein(mean±SEM, n=5). *pb0.05.


Recently, Dulyaninova et al.36 could not recapitulatethe phenotype of CKII-phosphorylated myosin IIAby mutation of the CKII target residues to asparticacid. Thus, our aspartate mutants may give an un-derestimation of the effects of TRPM7-mediatedphosphorylation on myosin IIA filament stability.Although most point mutations in the MHCIIA tailproduce relatively modest effects on salt-assemblycharacteristics, these mutations can have a large im-pact on cell behavior and the pathogenesis of humandisease. Several (point) mutations that cause MYH9-related diseases have been identified in myosinIIA.41,42 These single amino acid substitutions arefound mainly in the coiled-coil domain. While mostof these mutations produce minor effects on myosinIIA solubility, they do effectively interfere with or-ganization of the monomers into filaments.40 Thus,we cannot exclude that TRPM7-mediated phosphor-ylation of mammalian nonmuscle myosin IIA has agreater impact on the organization of the filaments invivo than measured by its solubility in vitro. Accord-

ingly, the small increase in solubility of myosin IIAindicates that the majority of the 3×D mutant retainssome kind of filamentous organization. This pheno-type is reminiscent of that described byDulyaninovaet al.,36 who found that the S1934D mutant stillassociates with endogenous myosin IIA. Never-theless, we clearly demonstrate a distinct subcellulardistribution of the recombinant myosin IIA proteinsin line with previous reports.26,43 Our findings andthose of others indicate that future work is necessaryto understand exactly how phosphorylation affectsthe organization and turnover of myosin II filamentsin vivo.The importance of heavy chain phosphorylation to

myosin IIA filament stability remains controversial.Initial studies reported that myosin IIA filamentassembly was predominantly regulated by S100A4binding,whereasmyosin IIBwas regulated by heavychain phosphorylation.44,45 More recently, it wasdemonstrated that phosphorylation of the MHCIIAby CKII and PKC isoforms leads to filament disas-sembly in vitro.25 Moreover, phosphorylation of thePKC site in myosin IIA correlates with exocytosis,suggesting a role for MHCIIA phosphorylation inregulated secretion.46 Our results clearly demon-strate that phosphorylation of the MHCIIA affectsfilament assembly in vivo. Mutation of TRPM7 phos-phorylation sites to aspartic acid inMHCIIA reducesthe incorporation of myosin IIA into the actomyosincytoskeleton, leading to a relocalization of the pro-tein away from the cortex.Whilemyosins IIA and IIBare both regulated by heavy chain phosphorylation,important differences exist between the two iso-forms. For instance, PKC phosphorylates myosin IIAin the coiled-coil domain, whereas it phosphorylatesmyosin IIB in the nonhelical tail.44,47 Moreover, dif-ferent signaling pathways control the assembly ofeach myosin II isoform as the kinetics of myosins IIAand IIB phosphorylation upon agonist stimulation ofmammalian cells differ and separate members of thePKC family, specifically, phosphorylated myosinsIIA and IIB.26,27,45 We conclude that both myosinsIIA and IIB are regulated by heavy chain phosphor-ylation in vivo but that specific signaling pathwayscontrol the assembly of these different myosin IIisoforms.To date, we have observed no global change in

myosin IIA phosphorylation in response to TRPM7activation (Supplementary Fig. S4). This result is notsurprising since myosin IIA is constitutively phos-phorylated on multiple sites, making it difficult todetect changes in 32P incorporation of a single site. Infact, detecting the phosphorylation of a particularsite on myosin II generally requires the mutation ofseveral residues that are phosphorylated by otherkinases.27 Moreover, the TRPM7 kinase is low inabundance relative to other kinases. Based on thelow expression levels, we do not believe that TRPM7is responsible for the global regulation of myosin IIAdynamics in mammalian cells and that additionalmechanisms are in place for this purpose. Instead,we propose that it affectsmyosin IIA locally. Accord-ingly, TRPM7 is enriched in cell adhesion structures,

Fig. 10. Relocalization of YFP–MHCIIA 3×D mutant away from the cortex. (a) Representative images of COS7 cellsexpressing myosin IIA mutants. YFP–MHCIIA constructs were transiently expressed in COS7 cells. Subsequently, the actincytoskeleton was stained using Texas Red-conjugated phalloidin and cellular localization of YFP–MHCIIA and F-actin wasvisualized by fluorescence microscopy. (b) Cortical index measurement of the myosin IIA mutants. The cortical index for 10representative cells was measured as previously described. Data are presented as mean±SEM. *pb0.001.


including podosomes.28,48 Activation of TRPM7maygenerate local relaxation of the actomyosin cytoske-leton in close proximity to adhesion structures, lea-ding to remodeling of focal adhesions and podo-somes.28 Our current efforts are aimed at definingthe cellular context in which phosphorylation ofThr1800, Ser1803 and Ser1808 by TRPM7 has itsstrongest effects.Both TRPM7 and PKC phosphorylate the MHCIIA

α-helical tail. Since TRPM7 and PKC are activated byphospholipase C-mediated phosphatidylinositol-4,5-bisphosphate hydrolysis,29,49 it is possible that bothkinases synergize to regulate myosin IIA filamentformation. Notably, activation of TRPM7 and that ofPKC lead to the remodeling of adhesion structuresand de novo formation of podosomes, which requiresinhibition of myosin II activity.28,50 It will therefore beinteresting to investigate how the different kinasepathways cooperate to provide temporal and spatialregulation of myosin II filament stability and local-ization in cells. The development of phospho-specificantibodies against the TRPM7, PKC and CKII siteswill be essential to address this issue. Ultimately,antibodies that allow in situ detection of phospho-MHCIIA will provide further insight into the dyna-mics of the actomyosin cytoskeleton in mammaliancells.

In conclusion, we demonstrate that TRPM7 regu-lates myosin IIA filament stability and protein local-ization by phosphorylating the heavy chain. More-over, our results identify novel residues, Thr1800,Ser1803 and Ser1808, that regulate myosin IIA fila-ment assembly in vivo. Future experiments will beaimed at understanding the spatial and temporalorganization of signaling networks controllingMHCIIA phosphorylation in mammalian cells.

Materials and Methods

Constructs

The retroviral vector encoding the hemagglutinin (HA)-taggedWTandKDTRPM7 kinase domain (HA-TRPM7-C;amino acids 1158–1864) was previously described.28Mouse MHCIIA fragments (Fig. 1) were amplified by re-verse transcription PCR using RNA isolated fromN1E-115cells and cloned in-frame in pGEX-1N using BamHI andEcoRI sites. HumanMHCIIA fragments fused to GSTweregenerated as described for mouse MHCIIA constructs, butthe cDNA was amplified by PCR using the pTRE-GFP(green fluorescent protein)–MHCIIA construct (kind gift ofRobert Adelstein, National Institutes of Health, Bethesda,MD, USA) as template. The vector pTRE-GFP–MHCIIA

†http://msquant.sourceforge.net


contains a Tet-responsive element,33 which allows the in-ducible expression of GFP–MHCIIA in mammalian cells;however, in our hands, expression in different cell lineswas inefficient. Therefore, MHCIIA was excised usingHindIII and SpeI sites and cloned into pEYFP-C3, whichcontains a cytomegalovirus promoter. YFP–MHCIIA wasdigested with AflII and SpeI, Klenow filled and ligated togenerate theΔC170mutant. The construct encodingmyosinIIA rod in the pET28a vectorwas previously described.20 Allpoint mutations were introduced by site-directed mutagen-esis using a QuikChange mutagenesis kit (Stratagene). Allconstructs were verified by DNA sequencing.

Cell culture

RBL-2H3 and COS7 cells were cultured in Dulbecco'smodified Eagle's mediumwith 10% fetal calf serum. StableRBL-2H3 cell lines expressing WT and KD HA-TRPM7-Cwere generated by retroviral transduction.51 Cells wereselected by the addition of 0.8mg/ml of G418 to themedia,and the selectionwas completewithin 7 days. For transientprotein expression, COS7 cells were transfected usingeither the diethylaminoethyl–dextran method or FuGENE6 (Roche) according to the manufacturer's protocol.

GST fusion protein purification

GST fusion proteins were induced in BL21(DE3) Esche-richia coli by the addition of 200 μM IPTG and incubatingthe culture at 37 °C for 3 h. Bacteria were washed andsubsequently resuspended in ice-cold GST lysis buffer[50mMTris, pH 7.5, 300mMNaCl, 1.5mMMgCl2, 0.2mMethylenediaminetetraacetic acid (EDTA), 0.5 mM DTT, 1%Triton X-100 and protease inhibitors]. Bacteria were incu-bated on ice for 15 min in the presence of lysozyme(100 μg/ml) to degrade the cell wall. The bacteria werelysed by sonication, and insoluble material was removedby centrifugation. The GST fusion proteins were isolatedby incubating the supernatant with glutathione–Sepharosebeads for 2 h at 4 °C. The beads were washed in GST lysisbuffer, and GST fusion proteins were eluted in elutionbuffer (100mMTris, pH 8.0, 300mMNaCl, 20mM reducedglutathione and 1 mM DTT). Protein concentration wasmeasured using the Bradford assay. For long-term storage,proteins were frozen in the presence of 10% glycerol at−80 °C.

In vitro kinase assays

WTand KDHA-TRPM7-C were purified frommamma-lian cells by immunoprecipitation. Cells were lysed in ice-cold RIPA buffer (50 mM Tris, pH 7.5, 150 mMNaCl, 0.5%sodium deoxycholate, 0.1% SDS, 1% Triton X-100 andprotease inhibitors). Debris was removed by centrifuga-tion, and TRPM7 kinase was isolated by immunoprecipita-tion using anti-HA antibodies (12CA5) coupled to proteinG–Sepharose. TRPM7 immune complexes were washedand resuspended in IVK buffer (50 mM Hepes, pH 7.0,4 mM MnCl2 and 2 mM DTT) with 2 μg of GST fusionproteins. The kinase reactions were initiated by addingATP (100 μM cold ATP plus 5 μCi [γ-32P]ATP) and incu-bating at 30 °C for 30 min. Kinase reactions were stoppedby the addition of Laemmli buffer containing 40 mMEDTA. Samples were boiled and separated by SDS-PAGE,and phosphorylated proteins were detected by autoradio-graphy. Quantification was performed by phosphorima-ger analysis.

Edman sequencing and MALDI–TOF MS

Residues phosphorylated by TRPM7 in MHCIIA weredetermined by a combination of solid-phase Edmansequencing and MS. Mapping of phosphorylation sitesby Edman degradation sequencing and MS analysis of32P-labeled peptides were performed as previouslydescribed.52

Phosphopeptide analysis by LC-MS/MS

Tryptic and GluC digests of phosphorylated myosin IIAwere purified and desalted using C18 STAGE tips53 beforeanalysis by MS. Peptide identification experiments wereperformed using a nano-HPLC Agilent 1100 nanoflowsystem connected online to a 7-T LTQ-FT mass spectro-meter (Thermo Fisher, Bremen, Germany). Peptides wereseparated on 15-cm 100-μm ID PicoTip columns (NewObjective, Woburn, MA, USA) packed with 3-μmReprosil C18 beads (Dr. Maisch GmbH, Ammerbuch,Germany) using a 90-min gradient from 10% buffer B to40% buffer B (buffer B contains 80% acetonitrile in 0.5%acetic acid), with a flow rate of 300 nl/min. Peptideseluting from the column tip were electrosprayed directlyinto the mass spectrometer with a spray voltage of 2.1 kV.Data acquisition with the LTQ-FT was performed in adata-dependent mode to automatically switch betweenMS, MS2 and neutral loss-dependent MS3 acquisitions.Briefly, full-scan MS spectra of intact peptides (m/z 350–2000) with an automated gain control accumulationtarget value of 106 ions were acquired in the FT ioncyclotron resonance cell with a resolution of 50,000. Thefour most abundant ions were sequentially isolated andfragmented in the linear ion trap by applying collisionallyinduced dissociation using an accumulation target valueof 10,000 (capillary temperature=100 °C; normalizedcollision energy=30%). A dynamic exclusion of ionspreviously sequenced within 180 s was applied. Allunassigned charge states and singly charged ions wereexcluded from sequencing. A minimum of 50 counts wasrequired for MS2 selection. Data-dependent neutral lossscanning of phosphoric acid groups was enabled for eachMS2 spectrum among the three most intense fragmentions. RAW spectrum files were converted into a Mascotgeneric peak list by DTA Supercharger.† Accurate parentmasses of MS3 spectra were automatically generated byDTA Supercharger from the full FT MS scan. Peptidesand proteins were identified using the Mascot (MatrixScience) search engine version 2.0 to search a localversion of the human International Protein Indexdatabase version 3.05 supplemented with the sequencesof GST–myosin IIA fusion proteins. The settings forMascot searches were 10-ppm tolerance for the parentalpeptide and 0.5 Da for fragmentation spectra, a fixedcarbamidomethyl modification for cysteines, variablemodifications for oxidation of methionine, deamidationfor glutamine and asparagine and phosphorylation forserine, threonine and tyrosine. Verification and sitemapping of phosphorylated peptides were performedusing the posttranslational modification scoring algo-rithm of MSQuant†, according to the procedure of Olsenet al. for phosphopeptides.54 As a final verification step,peptides containing phosphorylation sites occurring onlyonce or twice were verified by manual inspection of theMS2 and MS3 spectra.

http://msquant.sourceforge.net


Myosin IIA filament assembly

WT and mutant myosin IIA rods were purified as pre-viously described.20 The assembly properties of the myo-sin IIA rods were characterized in the presence of magne-sium as described by Dulyaninova et al.25 The solubilitydata were plotted as a function of NaCl concentration andfit to the Hill equation in order to compare the midpoint ofthe curves for WT and mutant rods.

Cytoskeletal extraction

COS7 cells were transiently transfected with YFP–MHCIIA constructs 24 h prior to the experiment. Cellswere washed twice with ice-cold phosphate bufferedsaline (PBS) and lysed in cytoskeletal extraction buffer(50mMTris, pH 7.5, 50 mMNaCl, 5mMMgCl2, 1% TritonX-100, 1 mM PMSF, 2 μg/ml of aprotinin and 2 μg/ml ofleupeptin) for 10 min on ice. The lysates were centrifugedfor 10 min at 16,000g to remove any insoluble material.Laemmli buffer was added to the supernatant. The puri-fied cytoskeletons were first washed twice with cytoske-letal extraction buffer and proteins were solubilized inLaemmli buffer to solubilize the Triton-insoluble frac-tion. The proteins were separated by SDS-PAGE on 6%gel and transferred to nitrocellulose. YFP–MHCIIAproteins were detected using anti-GFP (1:1000; Roche)antibodies followed by IRDye 800CW-conjugated anti-mouse immunoglobulin G (1:5000; Westburg). The fluo-rescence intensity of each band was quantified withthe use of an Odyssey Infrared Imaging System (LI-CORBiosciences).

Microscopy

COS7 cells were seeded on glass coverslips and trans-fected with YFP–MHCIIA constructs. On the followingday, cellswere fixed in 3.7% formaldehyde/PBS for 10min,permeabilized with 0.1% Triton X-100/PBS for 3 min andblocked in 3% bovine serum albumin/PBS. Cells wereincubated with Texas Red-labeled phalloidin (1:200;Molecular Probes) for 45 min to detect F-actin. Cells werewashed three timeswith PBS between each step. Cellswereviewed using a Leica DMRA microscope with a ×63, 1.32-NA oil immersion lens. Cortical index was measured aspreviously described.26

Statistical analysis

Quantitative data are presented as mean±SEM. Thestatistical significance of differences between experimentalgroups was assessed with Student's t test. Differences inmeans were considered significant if pb0.05.

Acknowledgements

This work was supported by a FEBS Short-TermFellowship to K. C. and a grant from the DutchCancer Society to C. G. F., W. H.M. and F. N. v. L.Wethank Dr. Robert Adelstein (National Institutes ofHealth) for providing reagents and Niels Peterse forproviding technical assistance.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2008.02.057

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TRPM7 Regulates Myosin IIA Filament Stability and Protein Localization by Heavy Chain Phosphorylation