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• W3033485736 abstract "•Myosin light chain (RLC) is phosphorylated in tyrosine•Aberrant cell function by RLC deletion is not corrected by phospho-mimetic RLC•Phosphorylation of RLC in Y155 impairs formation of functional myosin hexamers•Phospho-Y155 RLC mainly appears at lamellipodia Active non-muscle myosin II (NMII) enables migratory cell polarization and controls dynamic cellular processes, such as focal adhesion formation and turnover and cell division. Filament assembly and force generation depend on NMII activation through the phosphorylation of Ser19 of the regulatory light chain (RLC). Here, we identify amino acid Tyr (Y) 155 of the RLC as a novel regulatory site that spatially controls NMII function. We show that Y155 is phosphorylated in vitro by the Tyr kinase domain of epidermal growth factor (EGF) receptor. In cells, phosphorylation of Y155, or its phospho-mimetic mutation (Glu), prevents the interaction of RLC with the myosin heavy chain (MHCII) to form functional NMII units. Conversely, Y155 mutation to a structurally similar but non-phosphorylatable amino acid (Phe) restores the more dynamic cellular functions of NMII, such as myosin filament formation and nascent adhesion assembly, but not those requiring stable actomyosin bundles, e.g., focal adhesion elongation or migratory front-back polarization. In live cells, phospho-Y155 RLC is prominently featured in protrusions, where it prevents NMII assembly. Our data indicate that Y155 phosphorylation constitutes a novel regulatory mechanism that contributes to the compartmentalization of NMII assembly and function in live cells. Active non-muscle myosin II (NMII) enables migratory cell polarization and controls dynamic cellular processes, such as focal adhesion formation and turnover and cell division. Filament assembly and force generation depend on NMII activation through the phosphorylation of Ser19 of the regulatory light chain (RLC). Here, we identify amino acid Tyr (Y) 155 of the RLC as a novel regulatory site that spatially controls NMII function. We show that Y155 is phosphorylated in vitro by the Tyr kinase domain of epidermal growth factor (EGF) receptor. In cells, phosphorylation of Y155, or its phospho-mimetic mutation (Glu), prevents the interaction of RLC with the myosin heavy chain (MHCII) to form functional NMII units. Conversely, Y155 mutation to a structurally similar but non-phosphorylatable amino acid (Phe) restores the more dynamic cellular functions of NMII, such as myosin filament formation and nascent adhesion assembly, but not those requiring stable actomyosin bundles, e.g., focal adhesion elongation or migratory front-back polarization. In live cells, phospho-Y155 RLC is prominently featured in protrusions, where it prevents NMII assembly. Our data indicate that Y155 phosphorylation constitutes a novel regulatory mechanism that contributes to the compartmentalization of NMII assembly and function in live cells. Non-muscle myosin II (NMII) is a motor protein that produces actin-associated intracellular forces in non-muscle cells [1Heissler S.M. Sellers J.R. Various themes of myosin regulation.J. Mol. Biol. 2016; 428: 1927-1946Crossref PubMed Scopus (54) Google Scholar]. It integrates converging biochemical and mechanical signals, converting them into mechanical work by displacing and crosslinking actin filaments. Its activation and assembly generate diverse actomyosin structures of different mechanical properties and stability, which control migratory polarization, cell division, and other motility-related cellular activities (reviewed in [2Aguilar-Cuenca R. Juanes-García A. Vicente-Manzanares M. Myosin II in mechanotransduction: master and commander of cell migration, morphogenesis, and cancer.Cell. Mol. Life Sci. 2014; 71: 479-492Crossref PubMed Scopus (65) Google Scholar, 3Ma X. Adelstein R.S. The role of vertebrate nonmuscle myosin II in development and human disease.Bioarchitecture. 2014; 4: 88-102Crossref PubMed Scopus (61) Google Scholar]). These actomyosin structures organize asymmetrically inside cells. Asymmetry is generated by spatially restricted activation of NMII, which begins with phosphorylation of the regulatory light chain (RLC) on S19 (reviewed in [4Ikebe M. Regulation of the function of mammalian myosin and its conformational change.Biochem. Biophys. Res. Commun. 2008; 369: 157-164Crossref PubMed Scopus (39) Google Scholar]). This event activates NMII and enables its self-association into bipolar filaments that may disassemble or evolve into higher order structures [5Dasbiswas K. Hu S. Schnorrer F. Safran S.A. Bershadsky A.D. Ordering of myosin II filaments driven by mechanical forces: experiments and theory.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2018; 373: 20170114Crossref PubMed Scopus (18) Google Scholar]. Integrins, growth factors, and chemoattractants elicit S19 phosphorylation with different stoichiometry and kinetic profiles [6Betapudi V. Rai V. Beach J.R. Egelhoff T. Novel regulation and dynamics of myosin II activation during epidermal wound responses.Exp. Cell Res. 2010; 316: 980-991Crossref PubMed Scopus (23) Google Scholar, 7Vicente-Manzanares M. Horwitz A.R. Myosin light chain mono- and di-phosphorylation differentially regulate adhesion and polarity in migrating cells.Biochem. Biophys. Res. Commun. 2010; 402: 537-542Crossref PubMed Scopus (38) Google Scholar, 8Vicente-Manzanares M. Cabrero J.R. Rey M. Pérez-Martínez M. Ursa A. Itoh K. Sánchez-Madrid F. A role for the Rho-p160 Rho coiled-coil kinase axis in the chemokine stromal cell-derived factor-1alpha-induced lymphocyte actomyosin and microtubular organization and chemotaxis.J. Immunol. 2002; 168: 400-410Crossref PubMed Scopus (88) Google Scholar]. Additional phosphorylation events on different residues of the RLC have diverse effects on the stability of the actomyosin bundles. Phosphorylation of RLC on T18 increases 2-fold the ATPase activity of NMII [9Kamisoyama H. Araki Y. Ikebe M. Mutagenesis of the phosphorylation site (serine 19) of smooth muscle myosin regulatory light chain and its effects on the properties of myosin.Biochemistry. 1994; 33: 840-847Crossref PubMed Scopus (67) Google Scholar, 10Ikebe M. Hartshorne D.J. Elzinga M. Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin.J. Biol. Chem. 1986; 261: 36-39Abstract Full Text PDF PubMed Google Scholar], defining the most stable subgroup of actomyosin filaments that delineate the trailing edge of polarized cells [7Vicente-Manzanares M. Horwitz A.R. Myosin light chain mono- and di-phosphorylation differentially regulate adhesion and polarity in migrating cells.Biochem. Biophys. Res. Commun. 2010; 402: 537-542Crossref PubMed Scopus (38) Google Scholar]. Additional regulatory sites include S1 and S2, which negatively regulate NMII upon phosphorylation by protein kinase C (PKC) [11Nishikawa M. Sellers J.R. Adelstein R.S. Hidaka H. Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase.J. Biol. Chem. 1984; 259: 8808-8814PubMed Google Scholar], inactivating NMII at the leading edge during mesenchymal chemotaxis [12Asokan S.B. Johnson H.E. Rahman A. King S.J. Rotty J.D. Lebedeva I.P. Haugh J.M. Bear J.E. Mesenchymal chemotaxis requires selective inactivation of myosin II at the leading edge via a noncanonical PLCγ/PKCα pathway.Dev. Cell. 2014; 31: 747-760Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. The second mechanism of sorting of actomyosin bundles in polarized cells depends on the intrinsic properties of NMII paralogs as defined by different myosin heavy chain II (MHCII) genes. Mammalian cells express up to three paralogs (NMII-A, -B, and -C), and the corresponding MHCII isoforms are encoded in different genes: Myh9 encodes MHCII-A; Myh10 encodes MHCII-B; and Myh14 encodes MHCII-C [13Vicente-Manzanares M. Ma X. Adelstein R.S. Horwitz A.R. Non-muscle myosin II takes centre stage in cell adhesion and migration.Nat. Rev. Mol. Cell Biol. 2009; 10: 778-790Crossref PubMed Scopus (1164) Google Scholar]. NMII-B defines the more stable actomyosin assemblies, and deletion of MHCII-B impairs front-back migratory polarity [14Vicente-Manzanares M. Koach M.A. Whitmore L. Lamers M.L. Horwitz A.F. Segregation and activation of myosin IIB creates a rear in migrating cells.J. Cell Biol. 2008; 183: 543-554Crossref PubMed Scopus (156) Google Scholar, 15Vicente-Manzanares M. Zareno J. Whitmore L. Choi C.K. Horwitz A.F. Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells.J. Cell Biol. 2007; 176: 573-580Crossref PubMed Scopus (287) Google Scholar]. Conversely, NMII-A is more dynamic [16Kovács M. Wang F. Hu A. Zhang Y. Sellers J.R. Functional divergence of human cytoplasmic myosin II: kinetic characterization of the non-muscle IIA isoform.J. Biol. Chem. 2003; 278: 38132-38140Crossref PubMed Scopus (173) Google Scholar], and its deletion impairs adhesion formation and compromises cell coherence [15Vicente-Manzanares M. Zareno J. Whitmore L. Choi C.K. Horwitz A.F. Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells.J. Cell Biol. 2007; 176: 573-580Crossref PubMed Scopus (287) Google Scholar, 17Cai Y. Biais N. Giannone G. Tanase M. Jiang G. Hofman J.M. Wiggins C.H. Silberzan P. Buguin A. Ladoux B. Sheetz M.P. Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow.Biophys. J. 2006; 91: 3907-3920Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar]. The stability of the NMII assemblies is also regulated by phosphorylation of specific residues in the tail domain of the heavy chain, including S1943 in MHCII-A [18Dulyaninova N.G. Malashkevich V.N. Almo S.C. Bresnick A.R. Regulation of myosin-IIA assembly and Mts1 binding by heavy chain phosphorylation.Biochemistry. 2005; 44: 6867-6876Crossref PubMed Scopus (112) Google Scholar], S1935 in MHCII-B [19Juanes-Garcia A. Chapman J.R. Aguilar-Cuenca R. Delgado-Arevalo C. Hodges J. Whitmore L.A. Shabanowitz J. Hunt D.F. Horwitz A.R. Vicente-Manzanares M. A regulatory motif in nonmuscle myosin II-B regulates its role in migratory front-back polarity.J. Cell Biol. 2015; 209: 23-32Crossref PubMed Scopus (24) Google Scholar], and other residues in MHCII-B and MHCII-C [20Even-Faitelson L. Ravid S. PAK1 and aPKCzeta regulate myosin II-B phosphorylation: a novel signaling pathway regulating filament assembly.Mol. Biol. Cell. 2006; 17: 2869-2881Crossref PubMed Scopus (72) Google Scholar, 21Clark K. Middelbeek J. Dorovkov M.V. Figdor C.G. Ryazanov A.G. Lasonder E. van Leeuwen F.N. The alpha-kinases TRPM6 and TRPM7, but not eEF-2 kinase, phosphorylate the assembly domain of myosin IIA, IIB and IIC.FEBS Lett. 2008; 582: 2993-2997Crossref PubMed Scopus (60) Google Scholar]. There are additional regulatory mechanisms of NMII function. For example, Tyr phosphorylation of MHCII motor domain affects NMII function, although the mechanism is poorly defined [22Almeida M.T. Mesquita F.S. Cruz R. Osório H. Custódio R. Brito C. Vingadassalom D. Martins M. Leong J.M. Holden D.W. et al.Src-dependent tyrosine phosphorylation of non-muscle myosin heavy chain-IIA restricts Listeria monocytogenes cellular infection.J. Biol. Chem. 2015; 290: 8383-8395Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 23Tsai R.K. Discher D.E. Inhibition of “self” engulfment through deactivation of myosin-II at the phagocytic synapse between human cells.J. Cell Biol. 2008; 180: 989-1003Crossref PubMed Scopus (248) Google Scholar]. On the other hand, Tyr phosphorylation of RLC was originally reported by Krebs and co-workers over 30 years ago [24Gallis B. Edelman A.M. Casnellie J.E. Krebs E.G. Epidermal growth factor stimulates tyrosine phosphorylation of the myosin regulatory light chain from smooth muscle.J. Biol. Chem. 1983; 258: 13089-13093PubMed Google Scholar], but its function in live cells has remained unexplored. Here, we find that replacing one specific Tyr residue, Y155, with a phospho-mimetic residue (Y→E) abrogates the association of the RLC to NMII. Y155 mutation prevents the formation of large and stable actomyosin bundles that define migratory polarity. When Y155 is phosphorylated by growth factor receptors, RLC is not incorporated into NMII hexamers. Likewise, RLC does not become phosphorylated on Y155 when associated to the NMII hexamer. RLC phosphorylated on Y155 is mainly localized at the leading edge of migrating cells. Together, these data reveal the existence of a novel mechanism that regulates NMII assembly and is crucial for the inhibition of NMII at the leading edge, driving the generation and maintenance of the intracellular gradients of assembled actomyosin that define front-rear polarity and adhesion dynamics in migrating cells. Tyr phosphorylation of the RLC on residues Y142 and Y155 in response to EGF was described early [24Gallis B. Edelman A.M. Casnellie J.E. Krebs E.G. Epidermal growth factor stimulates tyrosine phosphorylation of the myosin regulatory light chain from smooth muscle.J. Biol. Chem. 1983; 258: 13089-13093PubMed Google Scholar]. These phosphorylations were confirmed in several phospho-proteomics studies (https://www.phosphosite.org/; genes Myl9 and Myl12), but their biological significance remained unaddressed. To investigate this, we first expressed low levels of FLAG-RLC wild type (10%–20% the level of the endogenous protein) [14Vicente-Manzanares M. Koach M.A. Whitmore L. Lamers M.L. Horwitz A.F. Segregation and activation of myosin IIB creates a rear in migrating cells.J. Cell Biol. 2008; 183: 543-554Crossref PubMed Scopus (156) Google Scholar] in HEK293 cells and then treated the cells with peroxovanadate and calyculin A to inhibit Tyr and Ser/Thr phosphatases [25Webb D.J. Schroeder M.J. Brame C.J. Whitmore L. Shabanowitz J. Hunt D.F. Horwitz A.R. Paxillin phosphorylation sites mapped by mass spectrometry.J. Cell Sci. 2005; 118: 4925-4929Crossref PubMed Scopus (46) Google Scholar]. Subsequent mass spectrometric analysis of immunoprecipitated FLAG-RLC revealed that a substantial amount of RLC (≤10% total) was phosphorylated on Y155 (Figures 1A–1C) and on Y142 (Figures 1A, 1B, and 1D). As expected, S19 phosphorylation was readily detected (≈70% of total RLC), as well as a smaller percentage of bis-phosphorylated peptides, T18+S19 and Y142+Y155 (Figures 1A and 1B). In untreated conditions, Tyr phosphorylation was detectable but much lower (<1%). To study the significance of RLC Tyr phosphorylation in live cells, we generated RLC-GFP carrying phospho-mimetic (Y to E) and non-phosphorylatable (Y to F) mutations of Y142 and Y155. Low levels of mutant RLCs [14Vicente-Manzanares M. Koach M.A. Whitmore L. Lamers M.L. Horwitz A.F. Segregation and activation of myosin IIB creates a rear in migrating cells.J. Cell Biol. 2008; 183: 543-554Crossref PubMed Scopus (156) Google Scholar] were expressed in CHO.K1 cells, which were allowed to spread on fibronectin. None of the mutants affected the expression levels or localization of MHCII-A (Figure 2A). We found that RLC Y142F localized to myosin filaments similarly to wild-type RLC, whereas RLC Y142E localized there less frequently, often appearing in nuclei (Figures 2A and 2B). RLC Y155F behaved similarly to RLC Y142E. Finally, RLC Y155E almost never appeared in myosin filaments (Figures 2A and 2B), suggesting a decreased ability of RLC Y142E, Y155F, and particularly Y155E, to compete with wild-type RLC once contractile myosin filaments are formed. We next examined focal adhesion number, size, and distribution, which are proxies of contractility [26Dumbauld D.W. Shin H. Gallant N.D. Michael K.E. Radhakrishna H. García A.J. Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin-containing focal adhesions.J. Cell. Physiol. 2010; 223: 746-756PubMed Google Scholar]. Unexpectedly, cells expressing the RLC mutants displayed similar numbers of adhesions of comparable size (Figures 2A, 2C, and 2D). Next, we serum starved CHO.K1 cells expressing wild-type RLC or the RLC Y155 mutants, which reduced the numbers of NMII filaments and focal adhesions (Figures 2E–2G; compare to Figures 2A–2D). Upon stimulation with IGF-I for 3 h, cells expressing wild-type RLC displayed a marked increase in the number and area of focal adhesions (Figures 2E–2G). The number of adhesions also increased significantly in cells bearing the Y155F or Y155E mutant, although less than in wild-type, RLC-expressing cells (Figure 2F). However, the increase in total adhesive area in both mutants was very modest and not significant (Figure 2G). These experiments suggested that, in cells simultaneously expressing endogenous, wild-type RLC and the RLC Y155 mutants, the latter prevented the downstream signals through which IGF-I triggers NMII-dependent contraction from reaching endogenous RLC, effectively inhibiting focal adhesion elongation and maturation. The previous experiments suggested that residue Y155 plays a fundamental role in controlling NMII function in live cells. To further study this, we first decreased the interference of endogenous RLC by using a specific short hairpin RNA (shRNA) (Figure 3A). As previously published [27Park I. Han C. Jin S. Lee B. Choi H. Kwon J.T. Kim D. Kim J. Lifirsu E. Park W.J. et al.Myosin regulatory light chains are required to maintain the stability of myosin II and cellular integrity.Biochem. J. 2011; 434: 171-180Crossref PubMed Scopus (61) Google Scholar], RLC depletion also decreased the expression of MHCII-A and MHCII-B (Figures 3A and 3B), causing a spiky, round phenotype (Figure 3B). Expression of wild-type RLC and the Y142F, Y142E, and Y155F mutants to levels comparable to those of endogenous RLC (see STAR Methods) restored expression of MHCII-A (Figure 3B) and MHCII-B (Figure S1A). Conversely, Y155E did not (Figures 3B and S1A). RLC depletion had no effect on Myo18A, another myosin that interacts with RLC [28Guzik-Lendrum S. Heissler S.M. Billington N. Takagi Y. Yang Y. Knight P.J. Homsher E. Sellers J.R. Mammalian myosin-18A, a highly divergent myosin.J. Biol. Chem. 2013; 288: 9532-9548Crossref PubMed Scopus (45) Google Scholar, 29Tan I. Yong J. Dong J.M. Lim L. Leung T. A tripartite complex containing MRCK modulates lamellar actomyosin retrograde flow.Cell. 2008; 135: 123-136Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar] (Figures S1A and S1B). RLC depletion also abrogated focal adhesion formation (Figures 3B–3D; compare to Figures 2A, 2C, and 2D). Surprisingly, this effect extended to nascent adhesions in lamellipodia (Figure 3B, arrowheads in top-right panel). RLC wild type restored adhesion elongation and distribution (Figure 3B, arrows). Both Y142 mutants did as well, Y142F even better than wild-type RLC (Figures 3C and 3D). Importantly, Y155E-expressing, endogenous RLC-depleted cells remained devoid of elongated adhesions in the lamellum and cell body and nascent adhesions in the lamellipodium (Figure 3B, arrowheads and Figures 3C and 3D). Interestingly, RLC Y155F efficiently restored the number of adhesions (Figure 3C), including lamellipodial nascent adhesions (Figure 3B, arrows), but not their size (Figure 3D). We also observed that depletion of endogenous RLC improved the localization of RLC Y155F, but not Y155E, to myosin filaments (Figures 3B and 4A ). RLC depletion also increased cellular spreading and produced a significant loss of front-back polarity (Figures 4A and 4B). Expression of near-endogenous levels of wild-type RLC and both Y142 mutants also reversed this effect, but RLC Y155E did not. Interestingly, RLC Y155F did not restore polarity either, despite its ability to partially support NMII focal adhesion assembly and actomyosin filament formation (Figures 3C and 4A). RLC Y155F also displayed defective phosphorylation on S19 and T18+S19 in response to adhesion (Figure S2). Loss of front-rear polarity resulted in the inability of the cells to migrate properly, which was restored by expression of wild-type RLC, but not RLC Y155E (Figures 4C and 4D). RLC Y155F restored cell migration, although only partially (Figures 4C and 4D). NMII depletion or inhibition causes multinucleation [30Bao J. Jana S.S. Adelstein R.S. Vertebrate nonmuscle myosin II isoforms rescue small interfering RNA-induced defects in COS-7 cell cytokinesis.J. Biol. Chem. 2005; 280: 19594-19599Crossref PubMed Scopus (80) Google Scholar]. We recapitulated this by removing RLC. Multinucleation was reversed by expression of wild-type RLC and both Y142 mutants. Conversely, RLC Y155E did not reverse multinucleation. RLC Y155F reversed multinucleation but again only partially (Figure 4E). Together, these data delineate a key role for Y155 in the control of the cellular functions of NMII. The two most plausible mechanisms of action of Y155 phosphorylation are (1) Y155 phosphorylation controls the conformation of NMII and (2) it regulates the interaction of the RLC with MHCII as it incorporates into the NMII hexamer. To distinguish between the two possibilities, we first examined the interaction of the RLC Y155 mutants with MHCII (experiments targeting MHCII-B are shown; targeting MHCII-A yielded similar data) in control, live cells by co-immunoprecipitation. Immunoprecipitation of MHCII-B pulled down a reduced amount of RLC Y155F compared to wild-type RLC and a negligible amount of RLC Y155E (Figure 5A, second row). Reciprocal immunoprecipitation of RLC-GFP mutants revealed that MHCII-B was almost undetectable in RLC Y155E and reduced in RLC Y155F immunoprecipitates (Figure 5B, first row). These results suggested that Y155 phosphorylation negatively regulates the interaction of RLC with MHCII to form NMII hexamers. They also suggested that phospho-Y155 RLC does not interact properly with MHCII. However, Y155E RLC mimics phosphorylation, but it may not faithfully replicate all the effects of phosphorylation. To address this, we generated a polyclonal antibody (8471) that recognized phospho-Y155 RLC. The 8471 antibody recognized RLC (MW ≈ 20 kDa) in FLAG-transfected, peroxovanadate-treated CHO.K1 cells (Figure S3A). The antibody was deemed specific because it did recognize FLAG-tagged wild-type and Y142F-RLC, but not Y155F or a double mutant, YYFF (Figure S3A). Its intensity also decreased in peroxovanadate-treated, RLC-depleted cells with respect to non-depleted cells (Figure S3B). We next aimed at identifying the kinase(s) that could mediate Y155 phosphorylation. The Scansite algorithm [31Obenauer J.C. Cantley L.C. Yaffe M.B. Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs.Nucleic Acids Res. 2003; 31: 3635-3641Crossref PubMed Scopus (1294) Google Scholar] predicted that some receptors with tyrosine kinase activity (platelet-derived growth factor receptor [PDGFR] and epidermal growth factor receptor [EGFR]) could phosphorylate RLC directly on Y155. To test this, we produced recombinant, FLAG-tagged RLC (Figure 5C, third row) and used it as a substrate for a glutathione S-transferase (GST)-fusion protein containing the active kinase domain of EGFR (Figure 5C, first row). The kinase domain of EGFR robustly phosphorylated wild-type and Y142F, but not Y155F, RLC (Figure 5C, second row). We also addressed whether Y155 phosphorylation altered the conformation of the RLC. Using trypsin to cleave in vitro phosphorylated recombinant RLC, we found that phospho-Y155 RLC (and the Y155E mutant) displayed a modestly increased accessibility to proteolytic digestion (Figures S4A and S4B), suggesting that the conformation of phospho-Y155 RLC is slightly, but not dramatically, more accessible than that of non-phosphorylated RLC. We next immunoprecipitated MHCII-B from peroxovanadate-treated cells and examined the phosphorylation of NMII-B-bound RLC on Y155 and S19. As expected, we detected RLC phosphorylated on S19 (Figure 5D, third row). However, the immunoprecipitates did not contain phospho-Y155 RLC (Figure 5D, second row, right lanes), despite phospho-Y155 RLC being present in total lysates (Figure 5D, second row, left lanes). Together with the co-immunoprecipitation assays using RLC Y155E, these experiments suggest that, when phosphorylated on Y155, RLC interacts poorly with MHCII. To confirm this, we pulled down endogenous MHCII-B and incubated the immunoprecipitates with increasing amounts of FLAG-tagged wild type (or Y142F RLC to prevent interferences from the potential phosphorylation of Y142 by EGFR), non-phosphorylated or phosphorylated in Y155 by pre-incubation with active EGFR kinase and ATP. In both cases, non-phosphorylated RLC readily displaced endogenous RLC from immunoprecipitated MHCII-B, but Y155-phosphorylated RLC required a much higher concentration (Figure 5E, second and fourth rows and panels on right). This indicates that Y155-phosphorylated RLC displays lower affinity for MHCII-B than the non-phosphorylated form. These experiments prompted us to probe whether RLC could become phosphorylated on Y155 while bound to the NMII hexamer. In vitro phosphorylation experiments in which we used the entire NMII-B hexamer (immunoprecipitating MHCII-B) as a substrate for recombinant EGFR kinase revealed that, when in the context of the NMII-B hexamer, RLC does not become phosphorylated on Y155 (Figure 5F, second row, last lane), whereas it is readily phosphorylated in the NMII-unbound, free form (Figure 5F, left boxes). Also, experiments using recombinant subfragment-1 NMII-A produced in baculovirus transduced-Sf9 cells, or free recombinant His-tagged RLC followed by in vitro phosphorylation with recombinant EGFR kinase and mass spectrometry analysis yielded a similar conclusion (Figure S5). These data show that RLC becomes phosphorylated in Y155 when free, that is, outside of the context of the NMII hexamer, and that Y155 phosphorylation effectively prevents its incorporation to the hexamer. Growth factors induce actin-polymerization-driven protrusion. Others and we have shown that NMII is not actively assembling in the protruding region [32Cramer L.P. Siebert M. Mitchison T.J. Identification of novel graded polarity actin filament bundles in locomoting heart fibroblasts: implications for the generation of motile force.J. Cell Biol. 1997; 136: 1287-1305Crossref PubMed Scopus (240) Google Scholar, 33Vicente-Manzanares M. Newell-Litwa K. Bachir A.I. Whitmore L.A. Horwitz A.R. Myosin IIA/IIB restrict adhesive and protrusive signaling to generate front-back polarity in migrating cells.J. Cell Biol. 2011; 193: 381-396Crossref PubMed Scopus (100) Google Scholar]. Because growth factors promote Y155 phosphorylation in carcinoma cells [24Gallis B. Edelman A.M. Casnellie J.E. Krebs E.G. Epidermal growth factor stimulates tyrosine phosphorylation of the myosin regulatory light chain from smooth muscle.J. Biol. Chem. 1983; 258: 13089-13093PubMed Google Scholar], we sought to examine the potential role of phosphorylation of RLC on Y155 as a regulator of NMII assembly in the context of growth-factor-driven protrusion. CHO.K1 cells respond strongly to IGF-I [34Sunstrom N.A. Baig M. Cheng L. Payet Sugyiono D. Gray P. Recombinant insulin-like growth factor-I (IGF-I) production in super-CHO results in the expression of IGF-I receptor and IGF binding protein 3.Cytotechnology. 1998; 28: 91-100Crossref PubMed Google Scholar], eliciting many common downstream signals with EGF [35Voudouri K. Berdiaki A. Tzardi M. Tzanakakis G.N. Nikitovic D. Insulin-like growth factor and epidermal growth factor signaling in breast cancer cell growth: focus on endocrine resistant disease.Anal. Cell. Pathol. (Amst.). 2015; 2015: 975495PubMed Google Scholar]. In these cells, we observed a modest and sustained increase in Y155 phosphorylation in response to IGF-I (Figures 6A and 6B ). The kinetics of Y155 phosphorylation was different from those of S19, T18/S19, and S1, which reached a maximum after 30 min, declining thereafter (Figures 6A and 6B). Similar observations were made in A549 cells (Figure 6C) and in HEK293 cells in a dose-dependent manner in response to EGF (Figure 6D). These experiments indicate that growth factors of the EGF/insulin growth factor (IGF)-I family trigger phosphorylation of RLC on Y155 in live cells, albeit modestly. To investigate whether the increase in Y155 phosphorylation was spatially restricted, we examined the localization of phospho-Y155 RLC in fibronectin-bound CHO.K1 cells treated with IGF-I or Phorbol 12-myristate 13-acetate (PMA). The antibody was extensively validated for use in immunofluorescence (Figure S6). We observed that, in cells treated (10 min) with IGF-I or PMA, phospho-Y155 RLC appeared in lamellipodia, whereas it was largely absent from the lamellum (Figure 7A). These data indicate that phospho-Y155 RLC, which is unable to associate to NMII filaments, accumulates at protrusive regions of the cell. Together with the fact that RLC exchanges rapidly in and out of NMII filaments as shown by fluorescence recovery after photobleaching (FRAP) (up to 80% in 60 s [36Watanabe T. Hosoya H. Yonemura S. Regulation of myosin II dynamics by phosphorylation and dephosphorylation of its light chain in epithelial cells.Mol. Biol. Cell. 2007; 18: 605-616Crossref PubMed Scopus (104) Google Scholar], which is higher than MHCII-A) [15Vicente-Manzanares M. Zareno J. Whitmore L. Choi C.K. Horwitz A.F. Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells.J. Cell Biol. 2007; 176: 573-580Crossref PubMed Scopus (287) Google Scholar], these experiments are consistent with a model in which growth-factor-driven phosphorylation of RLC on Y155 at the leading edge locally abrogates the exchange of RLC in and out of NMII filaments, thereby preventing filament stabilization a" @default.
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• W3033485736 title "Tyrosine Phosphorylation of the Myosin Regulatory Light Chain Controls Non-muscle Myosin II Assembly and Function in Migrating Cells" @default.
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