Joan M. Taylor

Focal adhesion kinase: a regulator of focal adhesion dynamics and cell movement.

Engagement of integrin receptors with extracellular ligands gives rise to the formation of complex multiprotein structures that link the ECM to the cytoplasmic actin cytoskeleton. These adhesive complexes are dynamic, often heterogeneous structures, varying in size and organization. In motile cells, sites of adhesion within filopodia and lamellipodia are relatively small and transient and are referred to as ‘focal complexes,’ whereas adhesions underlying the body of the cell and localized to the ends of actin stress fibers are referred to as ‘focal adhesions’. Signal transduction through focal complexes and focal adhesions has been implicated in the regulation of a number of key cellular processes, including growth factor induced mitogenic signals, cell survival and cell locomotion. The formation and remodeling of focal contacts is a dynamic process under the regulation of protein tyrosine kinases and small GTPases of the Rho family. In this review, we consider the role of the focal complex associated protein tyrosine kinase, Focal Adhesion Kinase (FAK), in the regulation of cell movement with the emphasis on how FAK regulates the flow of signals from the ECM to the actin cytoskeleton.

An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase.

The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM). Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression. The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis. Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling. To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, we have used expression cloning to identify cDNAs that encode potential FAK-binding proteins. We report here the identification of a cDNA that encodes a new member of the GTPase-activating protein (GAP) family of GTPase regulators. This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42. Subcellular localization studies using Graf-transfected chicken embryo cells indicates that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions. Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver. Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase. We suggest that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPase that control steps in integrin-initiated signaling events.

Selective Expression of an Endogenous Inhibitor of FAK Regulates Proliferation and Migration of Vascular Smooth Muscle Cells

ABSTRACT Extracellular matrix signaling via integrin receptors is important for smooth muscle cell (SMC) differentiation during vasculogenesis and for phenotypic modulation of SMCs during atherosclerosis. We previously reported that the noncatalytic carboxyl-terminal protein binding domain of focal adhesion kinase (FAK) is expressed as a separate protein termed FAK-related nonkinase (FRNK) and that ectopic expression of FRNK can attenuate FAK activity and integrin-dependent signaling (A. Richardson and J. T. Parsons, Nature 380:538–540, 1996). Herein we report that in contrast to FAK, which is expressed ubiquitously, FRNK is expressed selectively in SMCs, with particularly high levels observed in conduit blood vessels. FRNK expression was low during embryonic development, was significantly upregulated in the postnatal period, and returned to low but detectable levels in adult tissues. FRNK expression was also dramatically upregulated following balloon-induced carotid artery injury. In cultured rat aortic smooth muscle cells, overexpression of FRNK attenuated platelet-derived growth factor (PDGF)-BB-induced migration and also dramatically inhibited [3H]thymidine incorporation upon stimulation with PDGF-BB or 10% serum. These effects were concomitant with a reduction in SMC proliferation. Taken together, these data indicate that FRNK acts as an endogenous inhibitor of FAK signaling in SMCs. Furthermore, increased FRNK expression following vascular injury or during development may alter the SMC phenotype by negatively regulating proliferative and migratory signals.

Sphingosine 1-phosphate stimulates smooth muscle cell differentiation and proliferation by activating separate serum response factor co-factors.

Sphingosine 1-phosphate (S1P) is a lipid agonist that regulates smooth muscle cell (SMC) and endothelial cell functions by activating several members of the S1P subfamily of G-protein-coupled Edg receptors. We have shown previously that SMC differentiation is regulated by RhoA-dependent activation of serum response factor (SRF). Because S1P is a strong activator of RhoA, we hypothesized that S1P would stimulate SMC differentiation. Treatment of primary rat aortic SMC cells with S1P activated RhoA as measured by precipitation with a glutathione S-transferase-rhotekin fusion protein. In SMC and 10T½ cells, S1P treatment up-regulated the activities of several transiently transfected SMC-specific promoters, and these effects were inhibited by the Rho-kinase inhibitor, Y-27632. S1P also increased smooth muscle α-actin protein levels in SMC but had no effect on SRF binding to the smooth muscle α-actin CArG B element. Quantitative reverse transcriptase-PCR showed that S1P treatment of SMC or 10T½ cells did not increase the mRNA level of either of the recently identified SRF co-factors, myocardin or myocardin-related transcription factor-A (MRTF-A). MRTF-A protein was expressed highly in SMC and 10T½ cultures, and importantly the effects of S1P were inhibited by a dominant negative form of MRTF-A indicating that S1P may regulate the transcriptional activity of MRTF-A. Indeed, S1P treatment increased the nuclear localization of FLAG-MRTF-A, and the effect of MRTF-A overexpression on smooth muscle α-actin promoter activity was inhibited by dominant negative RhoA. S1P also stimulated SMC growth by activating the early growth response gene, c-fos. This effect was not attenuated by Y-27632 but could be inhibited by the MEK inhibitor, UO126. S1P enhanced SMC growth through ERK-mediated phosphorylation of the SRF co-factor, Elk-1, as measured by gel shift and Elk-1 activation assays. Taken together these results demonstrate that S1P activates multiple signaling pathways in SMC and regulates proliferation by ERK-dependent activation of Elk-1 and differentiation by RhoA-dependent activation of MRTF-A.

Muscle ring finger protein-1 inhibits PKCΕ activation and prevents cardiomyocyte hypertrophy

Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy. In spite of this, we still know little about the mechanisms that inhibit hypertrophic growth. We define a novel anti-hypertrophic signaling pathway regulated by muscle ring finger protein-1 (MURF1) that inhibits the agonist-stimulated PKC-mediated signaling response in neonatal rat ventricular myocytes. MURF1 interacts with receptor for activated protein kinase C (RACK1) and colocalizes with RACK1 after activation with phenylephrine or PMA. Coincident with this agonist-stimulated interaction, MURF1 blocks PKCε translocation to focal adhesions, which is a critical event in the hypertrophic signaling cascade. MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1. MURF1 inhibits phenylephrine-induced (but not IGF-1–induced) increases in cell size. These findings establish that MURF1 is a key regulator of the PKC-dependent hypertrophic response and can blunt cardiomyocyte hypertrophy, which may have important implications in the pathophysiology of clinical cardiac hypertrophy.

Myocyte-Restricted Focal Adhesion Kinase Deletion Attenuates Pressure Overload–Induced Hypertrophy

Focal adhesion kinase (FAK) is a ubiquitously expressed cytoplasmic tyrosine kinase strongly activated by integrins and neurohumoral factors. Previous studies have shown that cardiac FAK activity is enhanced by hypertrophic stimuli before the onset of overt hypertrophy. Herein, we report that conditional deletion of FAK from the myocardium of adult mice did not affect basal cardiac performance, myocyte viability, or myofibrillar architecture. However, deletion of FAK abolished the increase in left ventricular posterior wall thickness, myocyte cross-sectional area, and hypertrophy-associated atrial natriuretic factor induction following pressure overload. Myocyte-restricted deletion of FAK attenuated the initial wave of extracellular signal-regulated kinase activation and cFos expression induced by adrenergic agonists and biomechanical stress. In addition, we found that persistent challenge of mice with myocyte-restricted FAK inactivation leads to enhanced cardiac fibrosis and cardiac dysfunction in comparison to challenged genetic controls. These studies show that loss of FAK impairs normal compensatory hypertrophic remodeling without a concomitant increase in apoptosis in response to cardiac pressure overload and highlight the possibility that FAK activation may be a common requirement for the initiation of this compensatory response.

The Histone Demethylase, Jmjd1a, Interacts With the Myocardin Factors to Regulate SMC Differentiation Marker Gene Expression

We and others have previously shown that the myocardin transcription factors play critical roles in the regulation of smooth muscle cell (SMC) differentiation marker gene expression. In a yeast 2-hybrid screen for proteins that interact with myocardin-related transcription factor-A (MRTF-A), we identified the histone 3 lysine 9 (H3K9)-specific demethylase, Jmjd1a. GST pull-down assays demonstrated that Jmjd1a bound all 3 myocardin family members, and further mapping studies showed that the jumonjiC domain of Jmjd1a was sufficient to mediate this interaction. Overexpression of Jmjd1a in multipotential 10T1/2 cells decreased global levels of di-methyl H3K9, stimulated the SM &agr;-actin and SM22 promoters, and synergistically enhanced MRTF-A- and myocardin-dependent transactivation. Using chromatin immunoprecipitation assays, we also demonstrated that TGF-&bgr;–mediated upregulation of SMC differentiation marker gene expression in 10T1/2 cells was associated with decreased H3K9 dimethylation at the CArG-containing regions of the SMC differentiation marker gene promoters. Importantly, knockdown of Jmjd1a in 10T1/2 cells and primary rat aortic SMCs by retroviral delivery of siRNA attenuated TGF-&bgr;–induced upregulation of endogenous SM myosin heavy chain expression. These effects were concomitant with increased H3K9 dimethylation at the SMC differentiation marker gene promoters and with inhibition of MRTF-A-dependent transactivation of the SMC-specific transcription. These results suggest, for the first time, that SMC differentiation marker gene expression is regulated by H3K9 methylation and that the effects of the myocardin factors on SMC-specific transcription may involve the recruitment of Jmjd1a to the SMC-specific promoters.

Conditional Deletion of Focal Adhesion Kinase Leads to Defects in Ventricular Septation and Outflow Tract Alignment

ABSTRACT To examine a role for focal adhesion kinase (FAK) in cardiac morphogenesis, we generated a line of mice with a conditional deletion of FAK in nkx2-5-expressing cells (herein termed FAKnk mice). FAKnk mice died shortly after birth, likely resulting from a profound subaortic ventricular septal defect and associated malalignment of the outflow tract. Additional less penetrant phenotypes included persistent truncus arteriosus and thickened valve leaflets. Thus, conditional inactivation of FAK in nkx2-5-expressing cells leads to the most common congenital heart defect that is also a subset of abnormalities associated with tetralogy of Fallot and the DiGeorge syndrome. No significant differences in proliferation or apoptosis between control and FAKnk hearts were observed. However, decreased myocardialization was observed for the conal ridges of the proximal outflow tract in FAKnk hearts. Interestingly, chemotaxis was significantly attenuated in isolated FAK-null cardiomyocytes in comparison to genetic controls, and these effects were concomitant with reduced tyrosine phosphorylation of Crk-associated substrate (CAS). Thus, it is possible that ventricular septation and appropriate outflow tract alignment is dependent, at least in part, upon FAK-dependent CAS activation and subsequent induction of polarized myocyte movement into the conal ridges. Future studies will be necessary to determine the precise contributions of the additional nkx2-5-derived lineages to the phenotypes observed.

Diaphanous 1 and 2 Regulate Smooth Muscle Cell Differentiation by Activating the Myocardin-Related Transcription Factors

Objective—We have previously shown that smooth muscle cell (SMC) differentiation marker gene expression is regulated by the small GTPase, RhoA. The objective of the present study was to determine the contributions of the RhoA effectors, diaphanous 1 and 2 (mDia1 and mDia2), to this regulatory mechanism. Methods and Results—mDia1 and mDia2 are expressed highly in aortic SMCs and in a number of SMC-containing organs including bladder, lung, and esophagus. Activation of mDia1/2 signaling by RhoA strongly stimulated SMC-specific promoter activity in multiple cell-types including primary aortic SMCs, and stimulated endogenous SM α-actin expression in 10T1/2 cells. Expression of a dominant negative Dia1 variant that inhibits both mDia1 and mDia2 significantly decreased SMC-specific transcription in SMCs. The effects of mDia1 and mDia2 required the presence of SRF and the activity of the myocardin transcription factors and were dependent on changes in actin polymerization. Importantly, stimulation of mDia1/2 signaling synergistically enhanced the activities of the myocardin-related transcription factors, MRTF-A and MRTF-B, and this effect was attributable to increased nuclear localization of these factors. Conclusions—These results indicate that RhoA-dependent signaling through mDia1/2 and the MRTFs is important for SMC-specific gene expression in SMCs.

Sphingosine 1-Phosphate Receptor 2 Signals Through Leukemia-Associated RhoGEF (LARG), to Promote Smooth Muscle Cell Differentiation

Objective—The goals of this study were to identify the signaling pathway by which sphingosine 1-phosphate (S1P) activates RhoA in smooth muscle cells (SMC) and to evaluate the contribution of this pathway to the regulation of SMC phenotype. Methods and Results—Using a combination of receptor-specific agonists and antagonists we identified S1P receptor 2 (S1PR2) as the major S1P receptor subtype that regulates SMC differentiation marker gene expression. Based on the known coupling properties of S1PR2 and our demonstration that overexpression of G&agr;12 or G&agr;13 increased SMC-specific promoter activity, we next tested whether the effects of S1P in SMC were mediated by the regulator of G protein-signaling-Rho guanine exchange factors (RGS-RhoGEFs) (leukemia-associated RhoGEF [LARG], PDZ-RhoGEF [PRG], RhoGEF [p115]). Although each of the RGS-RhoGEFs enhanced actin polymerization, myocardin-related transcription factor-A nuclear localization, and SMC-specific promoter activity when overexpressed in 10T1/2 cells, LARG exhibited the most robust effect and was the only RGS-RhoGEF activated by S1P in SMC. Importantly, siRNA-mediated depletion of LARG significantly inhibited the activation of RhoA and SMC differentiation marker gene expression by S1P. Knockdown of LARG had no effect on SMC proliferation but promoted SMC migration as measured by scratch wound and transwell assays. Conclusion—These data indicate that S1PR2-dependent activation of RhoA in SMC is mediated by LARG and that this signaling mechanism promotes the differentiated SMC phenotype.