Myosin II activity is not essential for recruitment of myosin II to the furrow in dividing HeLa cells

To elucidate whether phosphorylation of myosin II regulatory light chain (MRLC) is essential for myosin II recruitment to the furrow during cytokinesis, HeLa cells transfected with three types of GFP-tagged recombinant MRLCs, wild-type MRLC, non-phosphorylated form of MRLC, and phosphorylated form of MRLC, were examined. Living cell-imaging showed that both phosphorylated and non-phosphorylated form of MRLCs were recruited to the equator at the same time after anaphase onset, suggesting that phosphorylation of MRLC is not responsible for recruitment of myosin II to the equator. Moreover, the treatment with an inhibitor of myosin II activity, blebbistatin, induced no effect on recruitment of those three recombinant MRLCs. During cytokinesis, phosphorylated but not non-phosphorylated form of MRLC was retained in the equator. These results suggest that phosphorylation of MRLC is essential for retainment of myosin II in the furrow but not for initial recruitment of myosin II to the furrow in dividing HeLa cells.     

Enhancement of myosin II/actin turnover at the contractile ring induces slower furrowing in dividing HeLa cells

Myosin II ATPase activity is enhanced by the phosphorylation of MRLC (myosin II regulatory light chain) in non-muscle cells. It is well known that pMRLC (phosphorylated MRLC) co-localizes with F-actin (filamentous actin) in the CR (contractile ring) of dividing cells. Recently, we reported that HeLa cells expressing non-phosphorylatable MRLC show a delay in the speed of furrow ingression, suggesting that pMRLC plays an important role in the control of furrow ingression. However, it is still unclear how pMRLC regulates myosin II and F-actin at the CR to control furrow ingression during cytokinesis. In the present study, to clarify the roles of pMRLC, we measured the turnover of myosin II and actin at the CR in dividing HeLa cells expressing fluorescent-tagged MRLCs and actin by FRAP (fluorescence recovery after photobleaching). A myosin II inhibitor, blebbistatin, caused an enhancement of the turnover of MRLC and actin at the CR, which induced a delay in furrow ingression. Furthermore, only non-phosphorylatable MRLC and a Rho-kinase inhibitor, Y-27632, accelerated the turnover of MRLC and actin at the CR. Interestingly, the effect of Y-27632 was cancelled in the cell expressing phosphomimic MRLCs. Taken together, these results reveal that pMRLC reduces the turnover of myosin II and also actin at the CR. In conclusion, we show that the enhancement of myosin II and actin turnover at the CR induced slower furrowing in dividing HeLa cells.     

Diphosphorylated MRLC is required for organization of stress fibers in interphase cells and the contractile ring in dividing cells

Activity of nonmuscle myosin II is regulated by phosphorylation of its regulatory light chain (MRLC). Phosphoryration of MRLC at both Thr18 and Ser19 (diphosphorylation) results in higher MgATPase activity and in promotion of the assembly of myosin II filaments than does that of MRLC at Ser19 (monophosphorylation) in vitro. To determine the roles of the diphosphorylated MRLC in vivo, we transfected three kinds of MRLC mutants, unphosphorylated, monophosphorylated and diphosphorylated forms (MRLC2(T18AS19A), substitution of both Ser19 and Thr18 by Ala; MRLC2(T18AS19D), Ser19 by Asp and Thr18 by Ala; and MRLC2(T18DS19D), both Ser19 and Thr18 by Asp, respectively), into HeLa cells. Cells overexpressing the mutant MRLC2(T18DS19D) contained a larger number of actin filament bundles than did those overexpressing the mutant MRLC2(T18AS19D). Moreover, cells overexpressing the nonphosphorylatable mutant MRLC2(T18AS19A) showed a decrease in the number of actin filament bundles. Taken together, our data suggest that diphosphorylation of MRLC plays an important role in regulating actin filament assembly and reorganization in nonmuscle cells.     

Diphosphorylated but not monophosphorylated myosin II regulatory light chain localizes to the midzone without its heavy chain during cytokinesis

Myosin II is activated by the monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). Its ATPase activity is further enhanced by MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC). As these phosphorylated MRLCs are colocalized with their heavy chains at the contractile ring in dividing cells, we believe that the phosphorylated MRLC acts as a subunit of the activated myosin II during cytokinesis. However, the distinct role(s) of 1P- and 2P-MRLC during cytokinesis has not been elucidated. In this study, a monoclonal antibody (4F12) specific for 2P-MRLC was raised and used to examine the roles of 2P-MRLC in cultured mammalian cells. Our confocal microscopic observations using 4F12 revealed that 2P-MRLC localized to the contractile ring, and, unexpectedly, to the midzone also. Interestingly, 2P-MRLC did not colocalize with 1P-MRLC, myosin II heavy chain, and F-actin at the midzone. These results suggest that 2P-MRLC has a role different from that of 1P-MRLC at the midzone, and is not a subunit of myosin II.     

Myosin II regulatory light chain as a novel substrate for AIM-1, an aurora/Ipl1p-related kinase from rat

Previous studies demonstrated that the phosphorylated myosin II regulatory light chain (MRLC) is localized at the cleavage furrow of dividing cells, suggesting that phosphorylation of MRLC plays an important role in cytokinesis. However, it remains unclear which kinase(s) phosphorylate MRLC during cytokinesis. AIM-1, an Aurora/Ipl1p-related kinase from rat, is known as a serine/threonine kinase that is required for cytokinesis. Here we examined the possibility that AIM-1 is a candidate for a kinase that phosphorylates MRLC during cytokinesis. As a result, we showed that AIM-1 monophosphorylated MRLC at Ser19 using two-dimensional phosphopeptide mapping analysis and several MRLC mutants. Furthermore, AIM-1 was colocalized with monophosphorylated MRLC at the cleavage furrow of dividing cells. We propose here that AIM-1 may participate in monophosphorylation of MRLC during cytokinesis.     

Distinct tissue distributions and subcellular localizations of differently phosphorylated forms of the myosin regulatory light chain in Drosophila

Nonmuscle myosin II (myosin hereafter) has well-established roles in generating contractile force on actin filaments during morphogenetic processes in all metazoans. Myosin activation is regulated by phosphorylation of the myosin regulatory light chain (MRLC, encoded by spaghettisquash or sqh in Drosophila) first on Ser21 and subsequently on Thr20. These phosphorylation events are positively controlled by a variety of kinases including myosin light chain kinase, Rho kinase, citron kinase, and AMP kinase and are negatively regulated by myosin phosphatase. The activation of myosin is thus highly regulated and likely developmentally controlled. In order to monitor the activity of myosin during development, we have generated antibodies against the monophosphorylated (Sqh1P) and diphosphorylated (Sqh2P) forms of Sqh. We first show that the antibodies are highly specific. We then used these antibodies to monitor myosin activation in wild type Drosophila tissues. Interestingly, Sqh1P and Sqh2P show distinct patterns of expression in embryos. Sqh1P is expressed nearly ubiquitously and outlines cells consistent with a junctional localization, whereas Sqh2P is strongly expressed on the apical surfaces and in filopodia of tissues undergoing extensive cell shape change or cell movements including the invaginating fore- and hindgut, the invaginating tracheal system, the dorsal pouch and the dorsal most row of epidermal (DME) cells during dorsal closure. In imaginal discs, Sqh1P predominantly localizes in the adherens junction, whereas Sqh2P locates to the apical domain. These antibodies thus have the potential to be very useful in monitoring myosin activation for functional studies of morphogenesis in Drosophila.     

Retrograde flow and myosin II activity within the leading cell edge deliver F-actin to the lamella to seed the formation of graded polarity actomyosin II filament bundles in migrating fibroblasts

In migrating fibroblasts actomyosin II bundles are graded polarity (GP) bundles, a distinct organization to stress fibers. GP bundles are important for powering cell migration, yet have an unknown mechanism of formation. Electron microscopy and the fate of photobleached marks show actin filaments undergoing retrograde flow in filopodia, and the lamellipodium are structurally and dynamically linked with stationary GP bundles within the lamella. An individual filopodium initially protrudes, but then becomes separated from the tip of the lamellipodium and seeds the formation of a new GP bundle within the lamella. In individual live cells expressing both GFP-myosin II and RFP-actin, myosin II puncta localize to the base of an individual filopodium an average 28 s before the filopodium seeds the formation of a new GP bundle. Associated myosin II is stationary with respect to the substratum in new GP bundles. Inhibition of myosin II motor activity in live cells blocks appearance of new GP bundles in the lamella, without inhibition of cell protrusion in the same timescale. We conclude retrograde F-actin flow and myosin II activity within the leading cell edge delivers F-actin to the lamella to seed the formation of new GP bundles.     

The phosphorylation state of MRLC is polyamine dependent in intestinal epithelial cells

Cell migration is important to the integrity of the gastrointestinal tract for the normal movement of cells from crypt to villi and the healing of wounds. Polyamines are essential to cell migration, mucosal restitution, and, hence, healing. Polyamine depletion by α-difluoromethyl ornithine (DFMO) inhibited migration by decreasing lamellipodia and stress fiber formation and preventing the activation of Rho-GTPases. Polyamine depletion increased the association of the thick F-actin cortex with phosphorylated myosin regulatory light chain (pMRLC). In this study, we determined why MRLC is constitutively phosphorylated as part of the actin cortex. Inhibition of myosin light chain kinase (MLCK) decreased RhoA and Rac1 activities and significantly inhibited migration. Polyamine depletion increased phosphorylation of MRLC (Thr18/Ser19) and stabilized the actin cortex and focal adhesions. The Rho-kinase inhibitor Y27632 increased spreading and migration by decreasing the phosphorylation of MRLC, remodeling focal adhesions, and by activating Rho-GTPases. Thus phosphorylation of MRLC appears to be the rate-limiting step during the migration of IEC-6 cells. In addition, increased localization of RhoA with the actin cortex in polyamine-depleted cells appears to activate Rho-kinase. In the absence of polyamines, activated Rho-kinase phosphorylates myosin phosphatase targeting subunit 1 (MYPT1) at serine-668 leading to its inactivation and preventing the recruitment of phosphatase (protein phosphastase, PP1cδ) to the actomyosin cortex. In this condition, MRLC is constitutively phosphorylated and cycling does not occur. Thus activated myosin binds F-actin stress fibers and prevents focal adhesion turnover, Rho-GTPase activation, and the remodeling of the cytoskeleton required for migration.     

Drosophila myosin phosphatase and its role in dorsal closure

Myosin phosphatase negatively regulates nonmuscle myosin II through dephosphorylation of the myosin regulatory light chain (MRLC). Its regulatory myosin-binding subunit, MBS, is responsible for regulating the catalytic subunit in response to upstream signals and for determining the substrate specificity. DMBS, the Drosophila homolog of MBS, was identified to study the roles of myosin phosphatase in morphogenesis. The embryos defective for both maternal and zygotic DMBS demonstrated a failure in dorsal closure. In the mutant embryos, the defects were mainly confined to the leading edge cells which failed to fully elongate. Ectopic accumulation of phosphorylated MRLC was detected in lateral region of the leading edge cells, suggesting that the role of DMBS is to repress the activation of nonmuscle myosin II at the subcellular location for coordinated cell shape change. Aberrant accumulation of F-actin within the leading edge cells may correspond to the morphological aberrations of such cells. Similar defects were seen in embryos overexpressing Rho-kinase, suggesting that myosin phosphatase and Rho-kinase function antagonistically. The genetic interaction of DMBS with mutations in the components of the Rho signaling cascade also indicates that DMBS functions antagonistically to the Rho signal transduction pathway. The results indicate an important role for myosin phosphatase in morphogenesis.     

Activation of actin-activated MgATPase activity of myosin II by phosphorylation with MAPK-activated protein kinase-1b (RSK-2)

Regulatory light chain of myosin II (MRLC) was identified as a novel substrate of p90 ribosomal S6 kinase (RSK)-2, a Ser/Thr protein kinase which is phosphorylated and activated by mitogen-activated protein kinase (MAPK) in vitro and in vivo. Phosphopeptide map of MRLC phosphorylated by RSK-2 was identical to that by myosin light chain kinase (MLCK). Phosphoserine was recovered by the phosphoamino acid analysis of MRLC phosphorylated by RSK-2. Further, phosphorylation using recombinant glutathione S-transferase (GST) fusion proteins of HeLa MRLC2 revealed that RSK-2 phosphorylated wild-type MRLC2 (GST-wtMRLC2) but not its mutants GST-MRLC2(S19A) or GST-MRLC2(T18AS19A) (alanine substituted for Ser19 or both Ser19 and Thr18). These results revealed that RSK-2 phosphorylates MRLC at Ser19 as did MLCK. Phosphorylation of myosin II by RSK-2 resulted in activation of actin-activated MgATPase activity of myosin II. Interestingly, RSK-2 activity to phosphorylate MRLC was suppressed by phosphorylation with MAPK. RSK-2 might be a mediator that regulates myosin II activity through the MAPK cascade.     

Regulation of myosin II dynamics by phosphorylation and dephosphorylation of its light chain in epithelial cells

Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase.     

Sites phosphorylated in myosin light chain in contracting smooth muscle

Purified smooth muscle myosin light chain can be phosphorylated at multiple sites by myosin light chain kinase and protein kinase C. We have determined the sites phosphorylated on myosin light chain in intact bovine tracheal smooth muscle. Stimulation with 10 microM carbachol resulted in 66 +/- 5% monophosphorylated and 11 +/- 2% diphosphorylated myosin light chain after 1 min, and 47 +/- 4% monophosphorylated and 5 +/- 2% diphosphorylated myosin light chain after 30 min. Myosin heavy chain contained 0.06 +/- 0.01 mol of phosphate/mol of protein which did not change with carbachol. At both 1 and 30 min the monophosphorylated myosin light chain contained only phosphoserine whereas the diphosphorylated myosin light chain contained both phosphoserine and phosphothreonine. Two-dimensional peptide mapping of tryptic digests of monophosphorylated and diphosphorylated myosin light chain obtained from carbachol-stimulated tissue was similar to the peptide maps of purified light chain monophosphorylated and diphosphorylated, respectively, by myosin light chain kinase; these maps were distinct from the map obtained with tracheal light chain phosphorylated by protein kinase C. Phosphorylation of tracheal smooth muscle myosin light chain by myosin light chain kinase yields the tryptic phosphopeptide ATSNVFAMFDQSQIQEFK with S the phosphoserine in the monophosphorylated myosin light chain and TS the phosphotreonine and phosphoserine in the diphosphorylated myosin light chain. Thus, stimulation of tracheal smooth muscle with a high concentration of carbachol results in formation of both monophosphorylated and diphosphorylated myosin light chain although the amount of diphosphorylated light chain is substantially less than monophosphorylated light chain. In the intact muscle, myosin light chain is phosphorylated at sites corresponding to myosin light chain kinase phosphorylation.     

Diphosphorylation of the myosin regulatory light chain enhances the tension acting on stress fibers in fibroblasts

Regulation of the contractile force is crucial for cell migration, cell proliferation, and maintenance of cell morphology. Phosphorylation of the myosin II regulatory light chain (MRLC) is involved in these processes. To show whether the diphosphorylation of MRLC increases the tension acting on stress fibers, changes in the stiffness of fibroblasts expressing wild-type MRLC and a mutant type, which cannot be diphosphorylated, on treatment with lysophosphatidic acid (LPA) were examined by a mechanical-scanning probe microscope (M-SPM). The LPA treatment increased cellular stiffness in the wild-type MRLC expressing cells, while it had no effect on the mutated cells. Immunostaining showed that LPA stimulation induced the diphosphorylation of MRLC. These results suggest that the diphosphorylation of MRLC enhances the tension acting on stress fibers.     

Aurora B but Not Rho/MLCK Signaling Is Required for Localization of Diphosphorylated Myosin II Regulatory Light Chain to the Midzone in Cytokinesis

Non-muscle myosin II is stimulated by monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC) further enhances the ATPase activity of myosin II. Phosphorylated MRLCs localize to the contractile ring and regulate cytokinesis as subunits of activated myosin II. Recently, we reported that 2P-MRLC, but not 1P-MRLC, localizes to the midzone independently of myosin II heavy chain during cytokinesis in cultured mammalian cells. However, the mechanism underlying the distinct localization of 1P- and 2P-MRLC during cytokinesis is unknown. Here, we showed that depletion of the Rho signaling proteins MKLP1, MgcRacGAP, or ECT2 inhibited the localization of 1P-MRLC to the contractile ring but not the localization of 2P-MRLC to the midzone. In contrast, depleting or inhibiting a midzone-localizing kinase, Aurora B, perturbed the localization of 2P-MRLC to the midzone but not the localization of 1P-MRLC to the contractile ring. We did not observe any change in the localization of phosphorylated MRLC in myosin light-chain kinase (MLCK)-inhibited cells. Furrow regression was observed in Aurora B- and 2P-MRLC-inhibited cells but not in 1P-MRLC-perturbed dividing cells. Furthermore, Aurora B bound to 2P-MRLC in vitro and in vivo. These results suggest that Aurora B, but not Rho/MLCK signaling, is essential for the localization of 2P-MRLC to the midzone in dividing HeLa cells.     

Inhibition by Rho-kinase and protein kinase C of myosin phosphatase is involved in thrombin-induced shape change of megakaryocytic leukemia cell line UT-7/TPO

Thrombin induced a shape change of UT-7/TPO, a thrombopoietin-dependent human megakaryocytic cell line. Expression of myosin light chain (MLC) kinase was negligible in UT-7/TPO cells, while Rho-kinase and protein kinase C (PKC) were detected. Thrombin stimulated both monophosphorylation at Ser19 and diphosphorylation at Thr18 and Ser19 of 20 kDa MLC, as well as phosphorylation of myosin-binding subunit (MBS) and PKC-potentiated inhibitory phosphoprotein of myosin phosphatase (CPI). The Rho-kinase inhibitor Y-27632 [(+)-(R)-trans-(1-aminoethyl)-N-(4-phynidyl) cyclohexane-carboxamide dihydrochloride, monohydrade] strongly inhibited thrombin-induced shape change, MBS phosphorylation, and mono- and diphosphorylation of MLC. The PKC inhibitor GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) partially inhibited thrombin-induced shape change and MLC diphosphorylation even at the concentration that completely inhibited thrombin-induced CPI phosphorylation. In shape-changed UT-7/TPO cells induced by thrombin, phosphorylated MBS and CPI were colocalized with diphosphorylated MLC at pseudopods, whereas monophosphorylated MLC was mainly located in the cortical region. The accumulation of diphosphorylated MLC was blocked by preincubation with either Y-27632 or GF109203X. These results suggest that Rho-kinase is responsible for the induction of MLC phosphorylation in thrombin-induced shape change of UT-7/TPO cells and that myosin phosphatase inactivation through Rho-kinase-MBS and PKC-CPI pathways could be necessary for enhancement of MLC diphosphorylation which promote the pseudopod formation.     

Phosphorylation of myosin II regulatory light chain controls its accumulation, not that of actin, at the contractile ring in HeLa cells

During cytokinesis in eukaryotic cells, an actomyosin-based contractile ring (CR) is assembled along the equator of the cell. Myosin II ATPase activity is stimulated by the phosphorylation of the myosin II regulatory light chain (MRLC) in vitro, and phosphorylated MRLC localizes at the CR in various types of cells. Previous studies have determined that phosphorylated MRLC plays an important role in CR furrowing. However, the role of phosphorylated MRLC in CR assembly remains unknown. Here, we have used confocal microscopy to observe dividing HeLa cells expressing fluorescent protein-tagged MRLC mutants and actin during CR assembly near the cortex. Di-phosphomimic MRLC accumulated at the cell equator earlier than non-phosphorylatable MRLC and actin. Interestingly, perturbation of myosin II activity by non-phosphorylatable MRLC expression or treatment with blebbistatin, a myosin II inhibitor, did not alter the time of actin accumulation at the cell equator. Furthermore, inhibition of actin polymerization by treatment with latrunculin A had no effect on MRLC accumulation at the cell equator. Taken together, these data suggest that phosphorylated MRLC temporally controls its own accumulation, but not that of actin, in cultured mammalian cells.     

Epigallocatechin-3-O-gallate disrupts stress fibers and the contractile ring by reducing myosin regulatory light chain phosphorylation mediated through the target molecule 67 kDa laminin receptor

Epigallocatechin-3-O-gallate (EGCG), a major polyphenol of green tea, has been shown to inhibit the growth of various cancer cell lines. We show here that EGCG induced the disruption of stress fibers and decreased the phosphorylation of the myosin II regulatory light chain (MRLC) at Thr18/Ser19, which is necessary for both contractile ring formation and cell division. Indirect immunofluorescence analysis revealed that EGCG inhibited the concentration of both F-actin and the phosphorylated MRLC in the cleavage furrow at the equator of dividing cells. In addition, EGCG increased the percentages of cells in the G(2)/M phase and inhibited cell growth. Recently, we have demonstrated that the anticancer activity of EGCG is mediated by the metastasis-associated 67kDa laminin receptor (67LR). To explore whether the effect of EGCG is mediated by the 67LR, we transfected cells with short hairpin RNA (shRNA) expression vector to downregulate 67LR expression. When the 67LR was silenced, the suppressive effect of EGCG on the MRLC phosphorylation was significantly attenuated. These results suggest that EGCG inhibits the cell growth by reducing the MRLC phosphorylation and this effect is mediated by the 67LR.     

ZIP kinase is responsible for the phosphorylation of myosin II and necessary for cell motility in mammalian fibroblasts

Reorganization of actomyosin is an essential process for cell migration and myosin regulatory light chain (MLC20) phosphorylation plays a key role in this process. Here, we found that zipper-interacting protein (ZIP) kinase plays a predominant role in myosin II phosphorylation in mammalian fibroblasts. Using two phosphorylation site-specific antibodies, we demonstrated that a significant portion of the phosphorylated MLC20 is diphosphorylated and that the localization of mono- and diphosphorylated myosin is different from each other. The kinase responsible for the phosphorylation was ZIP kinase because (a) the kinase in the cell extracts phosphorylated Ser19 and Thr18 of MLC20 with similar potency; (b) immunodepletion of ZIP kinase from the cell extracts markedly diminished its myosin II kinase activity; and (c) disruption of ZIP kinase expression by RNA interference diminished myosin phosphorylation, and resulted in the defect of cell polarity and migration efficiency. These results suggest that ZIP kinase is critical for myosin phosphorylation and necessary for cell motile processes in mammalian fibroblasts.     

PKU-beta/TLK1 regulates myosin II activities, and is required for accurate equaled chromosome segregation

Tousled-like kinase 1 (or protein kinase ubiquitous, PKU-beta/TLK1) is a serine/threonine protein kinase that is implicated in chromatin remodeling, DNA replication and mitosis. RNAi-mediated PKU-beta/TLK1-depleted human cells showed aneuploidy, and immunofluorescence analysis of these cells revealed the unequal segregation of daughter chromosomes. Immunoblots indicated a substantial reduction in the phosphorylation level of Ser19/Thr18 on the myosin II regulatory light chain (MRLC) in PKU-beta/TLK1-depleted cells, with no change in total MRLC protein. To confirm the relationship between mitotic aberration and MRLC dysfunction, we expressed wild type MRLC or DD-MRLC (mimics diphosphorylation; substitution of both Thr18 and Ser19 with aspartate) in PKU-beta/TLK1-depleted cells. DD-MRLC expression dramatically reduced the unequal segregation of chromosomes. Our data suggest that human PKU-beta/TLK1 plays an important role in chromosome integrity via the regulation of myosin II dynamics by phosphorylating MRLC during mitosis.     

Compressive Stress Induces Dephosphorylation of the Myosin Regulatory Light Chain via RhoA Phosphorylation by the Adenylyl Cyclase/Protein Kinase A Signaling Pathway

Mechanical stress that arises due to deformation of the extracellular matrix (ECM) either stretches or compresses cells. The cellular response to stretching has been actively studied. For example, stretching induces phosphorylation of the myosin regulatory light chain (MRLC) via the RhoA/RhoA-associated protein kinase (ROCK) pathway, resulting in increased cellular tension. In contrast, the effects of compressive stress on cellular functions are not fully resolved. The mechanisms for sensing and differentially responding to stretching and compressive stress are not known. To address these questions, we investigated whether phosphorylation levels of MRLC were affected by compressive stress. Contrary to the response in stretching cells, MRLC was dephosphorylated 5 min after cells were subjected to compressive stress. Compressive loading induced activation of myosin phosphatase mediated via the dephosphorylation of myosin phosphatase targeting subunit 1 (Thr853). Because myosin phosphatase targeting subunit 1 (Thr853) is phosphorylated only by ROCK, compressive loading may have induced inactivation of ROCK. However, GTP-bound RhoA (active form) increased in response to compressive stress. The compression-induced activation of RhoA and inactivation of its effector ROCK are contradictory. This inconsistency was due to phosphorylation of RhoA (Ser188) that reduced affinity of RhoA to ROCK. Treatment with the inhibitor of protein kinase A that phosphorylates RhoA (Ser188) induced suppression of compression-stimulated MRLC dephosphorylation. Incidentally, stretching induced phosphorylation of MRLC, but did not affect phosphorylation levels of RhoA (Ser188). Together, our results suggest that RhoA phosphorylation is an important process for MRLC dephosphorylation by compressive loading, and for distinguishing between stretching and compressing cells.