Phosphorylation of caldesmon during smooth muscle contraction and cell migration or proliferation

Summary The actin-binding protein caldesmon (CaD) exists both in smooth muscle (the heavy isoform, h-CaD) and non-muscle cells (the light isoform, l-CaD). In smooth muscles h-CaD binds to myosin and actin simultaneously and modulates the actomyosin interaction. In non-muscle cells l-CaD binds to actin and stabilizes␣the actin stress fibers; it may also mediate the interaction between actin and non-muscle myosins. Both h- and l-CaD are phosphorylated in vivo upon stimulation. The major phosphorylation sites of h-CaD when activated by phorbol ester are the Erk-specific sites, modification of which is attenuated by the MEK inhibitor PD98059. The same sites in l-CaD are also phosphorylated when cells are stimulated to migrate, whereas in dividing cells l-CaD is phosphorylated more extensively, presumably by cdc2 kinase. Both Erk and cdc2 are members of the MAPK family. Thus it appears that CaD is a downstream effector of the Ras signaling pathways. Significantly, the phosphorylatable serine residues shared by both CaD isoforms are in the C-terminal region that also contains the actin-binding sites. Biochemical and structural studies indicated that phosphorylation of CaD at the Erk sites is accompanied by a conformational change that partially dissociates CaD from actin. Such a structural change in h-CaD exposes the myosin-binding sites on the actin surface and allows actomyosin interactions in smooth muscles. In the case of non-muscle cells, the change in l-CaD weakens the stability of the actin filament and facilitates its disassembly. Indeed, the level of l-CaD modification correlates very well in a reciprocal manner with the level of actin stress fibers. Since both cell migration and cell division require dynamic remodeling of actin cytoskeleton that leads to cell shape changes, phosphorylation of CaD may therefore serve as a plausible means to regulate these processes. Thus CaD not only links the smooth muscle contractility and non-muscle motility, but also provides a common mechanism for the regulation of cell migration and cell proliferation.     

钙调蛋白结合蛋白通过调节细胞骨架稳定性影响肿瘤细胞运动能力

轻链钙调蛋白结合蛋白（light-chain Caldesmon,l-CaD）是一种重要的肌动蛋白结合蛋白,普遍存在于众多非肌肉细胞中。体外研究证明,l-CaD能通过与肌动蛋白的结合起到促进原肌动蛋白（G-actin）聚合、稳定肌动蛋白纤维（F-actin）结构的作用。在磷酸化作用下,l-CaD能从肌动蛋白纤维上脱离并促进肌动蛋白纤维的解聚。该研究拟考察l-CaD在细胞内对细胞肌动蛋白骨架的调节作用,阐明l-CaD对细胞运动能力的影响,作者将天然低表达l-CaD的人源性乳腺癌细胞MCF-7作为细胞模型,在MCF-7胞内以基因转染的方式高表达外源野生型l-CaD及其磷酸化突变株A1234-CaD（不可磷酸化CaD）、D1234-CaD（完全磷酸化CaD）。首先,通过激光共聚焦扫描,探讨了l-CaD对细胞骨架重排的调节;其次,通过细胞迁移transwell阵列,检测了l-CaD对细胞迁移能力的影响;最后,在单细胞层次上测定了细胞基底牵张力、胰酶刺激下的细胞基底脱附能力,并进一步检测了l-CaD对细胞迁移子过程中细胞伸张、收缩的影响。研究结果显示,l-CaD在胞内对细胞骨架的形成有显著的调控作用。非磷酸化l-CaD主要富集在细胞骨架上,增强了细胞骨架的强度,导致细胞基底牵张力以及对胰酶的耐受性增强,但对细胞的迁移能力有显著的抑制作用;磷酸化l-CaD跟细胞骨架结合能力很弱,对细胞的运动能力没有显著影响。通过磷酸化,l-CaD起到了一个“蛋白开关”的作用,通过控制细胞骨架的解聚、重排来调节细胞的运动能力。     

The IQGAP Iqg1 is a regulatory target of CDK for cytokinesis in Candida albicans

Cyclin-dependent kinases (CDKs) drive and coordinate multiple cell-cycle events, including construction and contraction of the actomyosin ring during cytokinesis. However, it remains unclear whether CDKs regulate cytokinesis by directly targeting components of the ring. In a search for proteins containing consensus CDK phosphorylation sites in Candida albicans, we found that the IQGAP Iqg1 contains two dense clusters of 19 such sites flanking the actin-interacting CH domain. Here, we show that Iqg1 is indeed a phosphoprotein that undergoes cell-cycle-dependent phosphorylation and can be phosphorylated by purified Clb-Cdc28 kinases in vitro. Mass spectrometry identified several phosphoserine and phosphothreonine residues among these CDK sites. Mutating 15 of the CDK phosphorylation sites with alanine markedly reduced Iqg1 phosphorylation in vivo. The 15A mutation greatly stabilized Iqg1, caused both premature assembly and delayed disassembly of the actomyosin ring, blocked Iqg1 interaction with the actin-nucleating proteins Bni1 and Bnr1, and resulted in defects in cytokinesis. Our data therefore strongly support the idea that the Cdc28 CDK regulates cytokinesis partly by directly phosphorylating the actomyosin ring component Iqg1.     

The role of caldesmon in the regulation of endothelial cytoskeleton and migration

The actin- and myosin-binding protein, caldesmon (CaD) is an essential component of the cytoskeleton in smooth muscle and non-muscle cells and is involved in the regulation of cell contractility, division, and assembly of actin filaments. CaD is abundantly present in endothelial cells (EC); however, the contribution of CaD in endothelial cytoskeletal arrangement is unclear. To examine this contribution, we generated expression constructs of l-CaD cloned from bovine endothelium. Wild-type CaD (WT-CaD) and truncated mutants lacking either the N-terminal myosin-binding site or the C-terminal domain 4b (containing actin- and calmodulin-binding sites) were transfected into human pulmonary artery EC. Cell fractionation experiments and an actin overlay assay demonstrated that deleting domain 4b, but not the N-terminal myosin-binding site, resulted in decreased affinity to both the detergent-insoluble cytoskeleton and soluble actin. Recombinant WT-CaD co-localized with acto-myosin filaments in vivo, but neither of CaD mutants did. Thus both domain 4b and the myosin-binding site are essential for proper localization of CaD in EC. Overexpression of WT-CaD led to cell rounding and formation of a thick peripheral subcortical actin rim in quiescent EC, which correlated with decreased cellular migration. Pharmacological inhibition of p38 MAPK, but not ERK MAPK, caused disassembly of this peripheral actin rim in CaD-transfected cells and decreased CaD phosphorylation at Ser531 (Ser789 in human h-CaD). These results suggest that CaD is critically involved in the regulation of the actin cytoskeleton and migration in EC, and that p38 MAPK-mediated CaD phosphorylation may be involved in endothelial cytoskeletal remodeling.     

AIP1/WDR1 supports mitotic cell rounding

The actin cytoskeleton plays a fundamental role in configuring cell shapes and movements. Actin interacting protein 1 (AIP1)/tryptophan-aspartate-repeat protein 1 (WDR1) induces actin severing and disassembly cooperating with ADF/cofilin. We found that mitotic cell flattening but not rounding was manifested by suppression of AIP1/WDR1 in cells. This mitotic cell flattening was not due to any changes in phosphorylation and distribution of cofilin in cells. We carried out a direct observation of actin filament severing/disassembly assay and found that phosphorylated cofilin still somewhat severs/disassembles actin filaments and that AIP1/WDR1 effaces this in vitro. We suggest that the phosphorylation of ADF/cofilin will be insufficient to completely inhibit actin turnover during mitosis, and that AIP1/WDR1 could abort the severing/disassembly activity somewhat still carried out due to phosphorylated ADF/cofilin. This mechanism could be required to induce cell morphologic changes, especially mitotic cell rounding.     

Regulation of cell-matrix adhesion by OLA1, the Obg-like ATPase 1

Attachment of cells to the extracellular matrix induces clustering of membrane receptor integrins which in turn triggers the formation of focal adhesions (FAs). The adaptor/scaffold proteins in FAs provide linkage to actin cytoskeleton, whereas focal adhesion kinase (FAK) and other FA-associated kinases and phosphatases transduce integrin-mediated signaling cascades, promoting actin polymerization and progression of cell spreading. In this study, we explored the role of OLA1, a newly identified member of Obg-like ATPases, in regulating cell adhesion processes. We showed that in multiple human cell lines RNAi-mediated downregulation of OLA1 significantly accelerated cell adhesion and spreading, and conversely overexpression of OLA1 by gene transfection resulted in delayed cell adhesion and spreading. We further found that OLA1-deficient cells had elevated levels of FAK protein and decreased Ser3 phosphorylation of cofilin, an actin-binding protein and key regulator of actin filament dynamics, while OLA1-overexpressing cells exhibited the opposite molecular alterations in FAK and cofilin. These findings suggest that OLA1 plays an important negative role in cell adhesion and spreading, in part through the regulation of FAK expression and cofilin phosphorylation, and manipulation of OLA1 may lead to significant changes in cell adhesion and the associated phenotypes.     

Novel Phosphospecific Antibodies for Monitoring Phosphorylation of Proteins Encoded by the Myosin Light Chain Kinase Genetic Locus

Myosin light chain kinase (MLCK) and the kinase-related protein (KRP), also known as telokin, are the major independent protein products of the smooth muscle/non-muscle MLCK genetic locus. They share a common C-terminal part and major sites phosphorylated in vivo. Whereas MLCK is critically involved in myosin activation and contraction initiation in smooth muscle, KRP is thought to antagonize MLCK and to exert relaxation activity. Phosphorylation controls the MLCK and KRP activities. We generated two phosphorylation and site-specific antibodies to individually monitor levels of MLCK and KRP phosphorylation on critical sites. We quantified the level of KRP phosphorylation in smooth muscle before and after an increase in intracellular free Ca2+ and stimulation of adenylate cyclase, protein kinase C, and mitogen-activated protein kinases (MAP-kinases). Forskolin and phorbol-12,13-dibutyrate increased KRP phosphorylation at Ser13 from 25 to 100% but did not produce contraction in rat ileum. The level of Ser13 phosphorylation was not altered during Ca2+-dependent contraction evoked by KCl depolarization or carbachol, but subsequently increased to maximum during forskolin-induced relaxation. These data suggest that several intracellular signaling pathways control phosphorylation of KRP on Ser13 in smooth muscle and thus may contribute to relaxation. In contrast, phosphorylation level of Ser19 of KRP increased only slightly (from 30 to 40-45%) and only in response to MAP-kinase activation, arguing against its regulatory function in smooth muscle.     

EBP50 phosphorylation by Cdc2/Cyclin B kinase affects actin cytoskeleton reorganization and regulates functions of human breast cancer cell line MDA-MB-231

The actin cytoskeleton plays an important role in cell shape determination, adhesion and cell cycle progression. Ezrinradixin-moesin (ERM)-binding phosphoprotein 50 (EBP50), also known as Na⁺-H⁺ exchanger regulatory factor 1 (NHERF1), associates with actin cytoskeleton and is related to cell cycle progression. Its Ser279 and Ser301 residues are phosphorylated by cyclin-dependent kinase 2 (cdc2)/cyclin B during the mitosis phase. However, the biological significance of EBP50 phosphorylation mediated by cdc2/cyclin B is not clear. In the present study, MDA-MB-231 cells with low levels of endogenous EBP50 protein were stably transfected with constructs of EBP50 wild type (WT), phosphodeficient (serine 279 and serine 301 mutated to alanine-S279A/S301A) or phospho-mimetic (serine 279 and serine 301 mutated to aspartic acid-S279D/S301D) mutants. Subsequently, multiple phenotypes of these cells were characterized. Failure of cdc2/cyclin B-mediated EBP50 phosphorylation in cells expressing S279A/S301A (AA cells) significantly increased F-actin content, enhanced the adherence of cells to the extracellular matrix, altered cell morphology and caused defects in cytokinesis, as reflected in the formation of giant cells with heteroploid DNA and multinucleation or giant nuclei. Furthermore, knockdown of EBP50 expression in AA cells rescued cell defects such as the cytokinesis failure and abnormal cell morphology. EBP50 S279A/ S301A had a weaker binding affinity with actin than EBP50 S279D/S301D, which might explain the increase of F-actin content in the AA cells. The present results suggest that cdc2/cyclin B-mediated EBP50 phosphorylation may play a role in the regulation of various cell functions by affecting actin cytoskeleton reorganization.     

Phosphorylation of Serine 402 Regulates RacGAP Protein Activity of FilGAP Protein

FilGAP is a Rho GTPase-activating protein (GAP) that specifically regulates Rac. FilGAP is phosphorylated by ROCK, and this phosphorylation stimulates its RacGAP activity. However, it is unclear how phosphorylation regulates cellular functions and localization of FilGAP. We found that non-phosphorylatable FilGAP (ST/A) mutant is predominantly localized to the cytoskeleton along actin filaments and partially co-localized with vinculin around cell periphery, whereas phosphomimetic FilGAP (ST/D) mutant is diffusely cytoplasmic. Moreover, phosphorylated FilGAP detected by Phos-tag is also mainly localized in the cytoplasm. Of the six potential phosphorylation sites in FilGAP tested, only mutation of serine 402 to alanine (S402A) resulted in decreased cell spreading on fibronectin. FilGAP phosphorylated at Ser-402 is localized to the cytoplasm but not at the cytoskeleton. Although Ser-402 is highly phosphorylated in serum-starved quiescent cells, dephosphorylation of Ser-402 is accompanied with the cell spreading on fibronectin. Treatment of the cells expressing wild-type FilGAP with calyculin A, a Ser/Thr phosphatase inhibitor, suppressed cell spreading on fibronectin, whereas cells transfected with FilGAP S402A mutant were not affected by calyculin A. Expression of constitutively activate Arf6 Q67L mutant stimulated membrane blebbing activity of both non-phosphorylatable (ST/A) and phosphomimetic (ST/D) FilGAP mutants. Conversely, depletion of endogenous Arf6 suppressed membrane blebbing induced by FilGAP (ST/A) and (ST/D) mutants. Our study suggests that Arf6 and phosphorylation of FilGAP may regulate FilGAP, and phosphorylation of Ser-402 may play a role in the regulation of cell spreading on fibronectin.     

Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton

The actin cytoskeleton plays a fundamental role in all eukaryotic cells it is a major determinant of cell morphology and polarity and the assembly and disassembly of filamentous actin structures provides a driving force for dynamic processes such as cell motility, phagocytosis, growth cone guidance and cytokinesis. The ability to reorganize actin filaments is a fundamental property of embryonic cells during development; the shape changes accompanying gastrulation and dorsal closure, for example, are dependent on the plasticity of the actin cytoskeleton, while the ability of cells or cell extensions, such as axons, to migrate within the developing embryo requires rapid and spatially organized changes to the actin cytoskeleton in response to the external environment. Work in mammalian cells over the last decade has demonstrated the central role played by the highly conserved Rho family of small GTPases in signal transduction pathways that link plasma membrane receptors to the organization of the actin cytoskeleton.     

Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints

Functional analysis of a Bcl-xL phosphorylation mutant series has revealed that cells expressing Bcl-xL(Ser49Ala) mutant are less stable at G2 checkpoint after DNA damage and enter cytokinesis more slowly after microtubule poisoning, than cells expressing wild-type Bcl-xL. These effects of Bcl-xL(Ser49Ala) mutant seem to be separable from Bcl-xL function in apoptosis. Bcl-xL(Ser49) phosphorylation is cell cycle-dependent. In synchronized cells, phospho-Bcl-xL(Ser49) appears during the S phase and G2, whereas it disappears rapidly in early mitosis during prometaphase, metaphase and early anaphase, and re-appears during telophase and cytokinesis. During DNA damage-induced G2 arrest, an important pool of phospho-Bcl-xL(Ser49) accumulates in centrosomes which act as essential decision centers for progression from G2 to mitosis. During telophase/cytokinesis, phospho-Bcl-xL(Ser49) is found with dynein motor protein. In a series of in vitro kinase assays, specific small interfering RNA and pharmacological inhibition experiments, polo kinase 3 (PLK3) was implicated in Bcl-xL(Ser49) phosphorylation. These data indicate that, during G2 checkpoint, phospho-Bcl-xL(Ser49) is another downstream target of PLK3, acting to stabilize G2 arrest. Bcl-xL phosphorylation at Ser49 also correlates with essential PLK3 activity and function, enabling cytokinesis and mitotic exit.     

Mammal-specific, ERK-dependent, caldesmon phosphorylation in smooth muscle. Quantitation using novel anti-phosphopeptide antibodies.

Extracellular signal-regulated kinases (ERKs) phosphorylate the high molecular mass isoform of the actin-binding protein caldesmon (h-CaD) at two sites (Ser(759) and Ser(789)) during smooth muscle stimulation. To investigate the role of phosphorylation at these sites, antibodies were generated against phosphopeptides analogous to the sequences around Ser(759) and Ser(789). Affinity-purified antibodies were phosho- and sequence-specific. The major site of phosphorylation in h-CaD in porcine carotid arterial muscle strips was at Ser(789); however, the amount of phosphate did not vary appreciably with either KCl or phorbol ester stimulation. Phosphorylation at Ser(759) of h-CaD was almost undetectable (<0.005 mol of phosphate/mol of protein). Moreover, phosphorylation of the low molecular mass isoform of the protein (l-CaD) at the site analogous to Ser(789) was greater in serum-stimulated cultured smooth muscle cells than in serum-starved cells. Serum-stimulated l-CaD phosphorylation was attenuated by the protein kinase inhibitor PD98059. These data 1) identify Ser(789) of h-CaD as the major site of ERK-dependent phosphorylation in carotid arteries; 2) show that the level of phosphorylation at Ser(789) is relatively constant following carotid arterial muscle stimulation, despite an increase in total protein phosphate content; and 3) suggest a functional role for ERK-dependent l-CaD phosphorylation in cell division.     

Redox regulation of beta-actin during integrin-mediated cell adhesion

Redox sensitivity of actin toward an exogenous oxidative stress has recently been reported. We report here the first evidence of in vivo actin redox regulation by a physiological source of reactive oxygen species, specifically those species generated by integrin receptors during cell adhesion. Actin oxidation takes place via the formation of a mixed disulfide between cysteine 374 and glutathione; this modification is essential for spreading and for cytoskeleton organization. Impairment of actin glutathionylation, either through GSH depletion or expression of the C374A redox-insensitive mutant, greatly affects cell spreading and the formation of stress fibers, leading to inhibition of the disassembly of the actinomyosin complex. These data suggest that actin glutathionylation is essential for cell spreading and cytoskeleton organization and that it plays a key role in disassembly of actinomyosin complex during cell adhesion.     

Myosin heavy-chain kinase A from Dictyostelium possesses a novel actin-binding domain that cross-links actin filaments

Myosin heavy-chain kinase A (MHCK A) catalyses the disassembly of myosin II filaments in Dictyostelium cells via myosin II heavy-chain phosphorylation. MHCK A possesses a 'coiled-coil'-enriched domain that mediates the oligomerization, cellular localization and actin-binding activities of the kinase. F-actin (filamentous actin) binding by the coiled-coil domain leads to a 40-fold increase in MHCK A activity. In the present study we examined the actin-binding characteristics of the coiled-coil domain as a means of identifying mechanisms by which MHCK A-mediated disassembly of myosin II filaments can be regulated in the cell. Co-sedimentation assays revealed that the coiled-coil domain of MHCK A binds co-operatively to F-actin with an apparent K(D) of approx. 0.5 muM and a stoichiometry of approx. 5:1 [actin/C(1-498)]. Further analyses indicate that the coiled-coil domain binds along the length of the actin filament and possesses at least two actin-binding regions. Quite surprisingly, we found that the coiled-coil domain cross-links actin filaments into bundles, indicating that MHCK A can affect the cytoskeleton in two important ways: (1) by driving myosin II-filament disassembly via myosin II heavy-chain phosphorylation, and (2) by cross-linking/bundling actin filaments. This discovery, along with other supporting data, suggests a model in which MHCK A-mediated bundling of actin filaments plays a central role in the recruitment and activation of the kinase at specific sites in the cell. Ultimately this provides a means for achieving the robust and highly localized disruption of myosin II filaments that facilitates polarized changes in cell shape during processes such as chemotaxis, cytokinesis and multicellular development.     

Caldesmon is a cytoskeletal target for PKC in endothelium

We have previously shown that treatment of bovine endothelial cell (EC) monolayers with phorbol myristate acetate (PMA) leads to the thinning of cortical actin ring and rearrangement of the cytoskeleton into a grid-like structure, concomitant with the loss of endothelial barrier function. In the current work, we focused on caldesmon, a cytoskeletal protein, regulating actomyosin interaction. We hypothesized that protein kinase C (PKC) activation by PMA leads to the changes in caldesmon properties such as phosphorylation and cellular localization. We demonstrate here that PMA induces both myosin and caldesmon redistribution from cortical ring into the grid-like network. However, the initial step of PMA-induced actin and myosin redistribution is not followed by caldesmon redistribution. Co-immunoprecipitation experiments revealed that short-term PMA (5 min) treatment leads to the weakening of caldesmon ability to bind actin and, to the lesser extent, myosin. Prolonged incubation (15-60 min) with PMA, however, strengthens caldesmon complexes with actin and myosin, which correlates with the grid-like actin network formation. PMA stimulation leads to an immediate increase in caldesmon Ser/Thr phosphorylation. This process occurs at sites distinct from the sites specific for ERK1/2 phosphorylation and correlates with caldesmon dissociation from the actomyosin complex. Inhibition of ERK-kinase MEK fails to abolish grid-like structure formation, although reducing PMA-induced weakening of the cortical actin ring, whereas inhibition of PKC reverses PMA-induced cytoskeletal rearrangement. Our results suggest that PKC-dependent phosphorylation of caldesmon is involved in PMA-mediated complex cytoskeletal changes leading to the EC barrier compromise.     

CD44-mediated elongated T cell spreading requires Pyk2 activation by Src family kinases, extracellular calcium, phospholipase C and phosphatidylinositol-3 kinase

The proline-rich tyrosine kinase 2, Pyk2, is a focal adhesion related kinase expressed in T cells that is tyrosine phosphorylated and activated by integrin, chemokine or T cell receptor stimulation. Ligation of the cell adhesion molecule CD44 also induces Pyk2 phosphorylation and T cell spreading, and this is negatively regulated by the protein tyrosine phosphatase CD45. Here, we identify the activation requirements for Pyk2 and demonstrate its requirement for CD44-mediated elongated T cell spreading. Upon CD44-mediated cell spreading, Pyk2 was recruited to CD44 clusters in both CD45⁺ and CD45⁻ T cells, yet was more strongly phosphorylated in T cells lacking CD45. In these cells, Pyk2 phosphorylation was dependent on Src family kinase activity and required actin polymerisation, phosphatidylinositol-3 kinase and phospholipase C activity as well as extracellular calcium. Inhibition of any of these events prevented Pyk2 phosphorylation and T cell spreading. Transfection of a truncated form of Pyk2 lacking the kinase domain, PRNK, inhibited CD44-mediated cell spreading, demonstrating an important role for Pyk2. However, inhibition of microtubule turnover by Taxol prevented elongated T cell spreading but did not affect Pyk2 phosphorylation, indicating that microtubule reorganisation is downstream, or independent, of Pyk2 phosphorylation. Together this demonstrates that multiple factors are required for CD44-induced Pyk2 activation, which plays a critical role in CD44-mediated elongated T cell spreading.     

A novel PKC-regulated mechanism controls CD44 ezrin association and directional cell motility

The dynamic assembly and disassembly of membrane cytoskeleton junctional complexes is critical in cell migration. Here we describe a novel phosphorylation mechanism that regulates the hyaluronan receptor CD44. In resting cells, CD44 is constitutively phosphorylated at a single serine residue, Ser325. After protein kinase C is activated, a switch in phosphorylation results in CD44 being phosphorylated solely at an alternative residue, Ser291. Using fluorescence resonance energy transfer (FRET) monitored by fluorescence lifetime imaging microscopy (FLIM) and chemotaxis assays we show that phosphorylation of Ser291 modulates the interaction between CD44 and the cytoskeletal linker protein ezrin in vivo, and that this phosphorylation is critical for CD44-dependent directional cell motility.     

Substrate Compliance versus Ligand Density in Cell on Gel Responses

Substrate stiffness is emerging as an important physical factor in the response of many cell types. In agreement with findings on other anchorage-dependent cell lineages, aortic smooth muscle cells are found to spread and organize their cytoskeleton and focal adhesions much more so on “rigid” glass or “stiff” gels than on “soft” gels. Whereas these cells generally show maximal spreading on intermediate collagen densities, the limited spreading on soft gels is surprisingly insensitive to adhesive ligand density. Bell-shaped cell spreading curves encompassing all substrates are modeled by simple functions that couple ligand density to substrate stiffness. Although smooth muscle cells spread minimally on soft gels regardless of collagen, GFP-actin gives a slight overexpression of total actin that can override the soft gel response and drive spreading; GFP and GFP-paxillin do not have the same effect. The GFP-actin cells invariably show an organized filamentous cytoskeleton and clearly indicate that the cytoskeleton is at least one structural node in a signaling network that can override spreading limits typically dictated by soft gels. Based on such results, we hypothesize a central structural role for the cytoskeleton in driving the membrane outward during spreading whereas adhesion reinforces the spreading.     

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca²⁺-dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.     

Phosphorylation of Ser 402 impedes phosphatase activity of slingshot 1

By using mass spectrometry, we have identified Ser 402 as a new phosphorylation site within the catalytic domain of human slingshot 1 (SSH1). Phosphorylation at this site inhibits substrate binding and, thus, phosphatase activity in vitro, resulting in enrichment of phosphorylated cofilin in monolayer cell culture. We further demonstrate that protein kinase D (PKD) is upstream from Ser 402 phosphorylation. Accordingly, expression of active PKD in Drosophila phenotypically mimics the loss of SSH activity by inducing accumulation of phosphorylated cofilin and filamentous actin. We thus identify a universal mechanism by which PKD controls SSH1 phosphatase activity.