Requirements for localization of p130cas to focal adhesions.

p130cas (Cas) is an adapter protein that has an SH3 domain followed by multiple SH2 binding motifs in the substrate domain. It also contains a tyrosine residue and a proline-rich sequence near the C terminus, which are the binding sites for the SH2 and SH3 domains of Src kinase, respectively. Cas was originally identified as a major tyrosine-phosphorylated protein in v-Crk- and v-Src-transformed cells. Subsequently, Cas was shown to be inducibly tyrosine phosphorylated upon integrin stimulation; it is therefore regarded as one of the focal adhesion proteins. Using an immunofluorescence study, we examined the subcellular localization of Cas and determined the regions required for its localization to focal adhesions. In nontransformed cells, Cas was localized predominantly to the cytoplasm and partially to focal adhesions. However, in 527F-c-Src-transformed cells, Cas was localized mainly to podosomes, where the focal adhesion proteins are assembled. The localization of Cas to focal adhesions was also observed in cells expressing the kinase-negative 527F/295M-c-Src. A series of analyses with deletion mutants expressed in various cells revealed that the SH3 domain of Cas is necessary for its localization to focal adhesions in nontransformed cells while both the SH3 domain and the C-terminal Src binding domain of Cas are required in 527F-c-Src-transformed cells and fibronectin-stimulated cells. In addition, the localization of Cas to focal adhesions was abolished in Src-negative cells. These results demonstrate that the SH3 domain of Cas and the association of Cas with Src kinase play a pivotal role in the localization of Cas to focal adhesions.     

Nmp4/CIZ对成骨细胞功能的调节作用

核基质蛋白4(nuclear matrix protein,Nmp4),亦称p130cas结合锌指蛋白(cas-interacting zinc finger protein,CIZ),是一种位于细胞核及粘附斑且具有核质穿梭功能的转录调控因子.体内实验证明,Nmp4/CIZ抑制骨形成活性进而抑制骨密度和骨量增加,而对骨吸收参数无显著影响.Nmp4/CIZ通过特异性结合成骨细胞Ⅰ型胶原α1链和基质金属蛋白酶启动子上游调控序列,调节其转录表达,促进骨转换.此外,Nmp4/CIZ抑制骨形态蛋白和甲状旁腺激素诱导的成骨细胞分化,抑制成骨细胞增殖并促进凋亡发生.Nmp4/CIZ参与调节成骨细胞力学-化学信号转导,其表达沉默可抑制尾悬吊诱导的小鼠骨量减少,并促进流体剪切力诱导的成骨细胞β-catenin信号途径.这些体内外实验证据表明,Nmp4/CIZ主要通过负调控机制发挥作用,提示这是一个潜在的骨丢失治疗药物靶点.     

Identification of p130Cas as a substrate of Yersinia YopH (Yop51), a bacterial protein tyrosine phosphatase that translocates into mammalian cells and targets focal adhesions.

A number of pathogenic bacteria utilize type III secretion pathways to translocate virulence proteins into host eukaryotic cells. We identified a host target of YopH, a protein tyrosine phosphatase that is translocated into mammalian cells by Yersiniae. A catalytically inactive 'substrate-trapping' mutant, YopHC403S, was used as a probe to determine where YopH substrates localize in eukaryotic cells. Immunofluorescence microscopy demonstrated that YopHC403S localized to focal adhesions in human epithelial cells infected with Y. pseudotuberculosis. YopHC403S stabilized focal adhesions, as shown by its dominant-negative effect on focal adhesion disassembly mediated by YopE, a translocated protein which disrupts actin stress fibers. Conversely, YopH destabilized focal adhesions, even in the absence of YopE, as shown by loss of phosphotyrosine staining. Immunoprecipitation revealed that YopHC403S was trapped in a complex with a hyperphosphorylated 125-135 kDa protein, identified by immunoblotting as the focal adhesion protein p130Cas. YopHC403S bound directly to p130Cas in a phosphotyrosine-dependent manner in vitro. Translocation of YopH into cells plated on fibronectin resulted in rapid and selective dephosphorylation of p130Cas. These results demonstrate that YopH targets focal adhesions in host cells and that p130Cas, a docking protein for multiple SH2 domains, is a direct substrate of this enzyme in vivo.     

Two substrate-targeting sites in the Yersinia protein tyrosine phosphatase co-operate to promote bacterial virulence

YopH is a protein tyrosine phosphatase and an essential virulence determinant of the pathogenic bacterium Yersinia. Yersinia delivers YopH into infected host cells using a type III secretion mechanism. YopH dephosphorylates several focal adhesion proteins including p130Cas in human epithelial cells, resulting in disruption of focal adhesions and cell detachment from the extracellular matrix. How the C-terminal protein tyrosine phosphatase domain of YopH targets specific substrates such as p130Cas in the complex milieu of the host cell has not been fully elucidated. An N-terminal non-catalytic domain of YopH binds p130Cas in a phosphotyrosine-dependent manner and functions as a novel substrate-targeting site. The structure of the YopH protein tyrosine phosphatase domain bound to a model phosphopeptide substrate was solved and the resulting structure revealed a second substrate-targeting site ('site 2') within the catalytic domain. Site 2 binds to p130Cas in a phosphotyrosine-dependent manner, and co-operates with the N-terminal domain ('site 1') to promote efficient recognition of p130Cas by YopH in epithelial cells. The identification of two substrate-targeting sites in YopH that co-operate to promote epithelial cell detachment and bacterial virulence reinforces the importance of protein-protein interactions for determining protein tyrosine phosphatase specificity in vivo, and highlights the sophisticated nature of microbial pathogenicity factors.     

P130Cas Src-binding and substrate domains have distinct roles in sustaining focal adhesion disassembly and promoting cell migration

The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas -/- mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif were able to partially restore the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly attempts. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation.     

Breast Cancer Anti-estrogen Resistance 3 (BCAR3) Protein Augments Binding of the c-Src SH3 Domain to Crk-associated Substrate (p130cas)

The focal adhesion adapter protein p130(cas) regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130(cas). AND-34/BCAR3, one of three NSP family members, binds the p130(cas) carboxyl terminus, adjacent to a bipartite p130(cas) Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130(cas). Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130(cas) complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130(cas) to bind the Src SH3 domain through an RPLPSPP motif in the p130(cas) SBD. Although our prior work identified phosphorylation of the serine within the p130(cas) RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130(cas). The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130(cas) complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130(cas) substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130(cas). Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130(cas) and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130(cas) complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130(cas) SBD.     

Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases.

Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. Among these are the focal adhesion proteins p130cas (Cas) and focal adhesion kinase (FAK). Here we identify the kinase(s) mediating integrin-induced Cas phosphorylation and characterize protein-protein interactions mediated by phosphorylated Cas. We found that expression of a constitutively active FAK in fibroblasts results in a consecutive tyrosine phosphorylation of Cas. This effect required the autophosphorylation site of FAK, which is a binding site for Src family kinases. Integrin-mediated phosphorylation of Cas was not, however, compromised in fibroblasts lacking FAK. In contrast, adhesion-induced tyrosine phosphorylation of Cas was reduced in cells lacking Src, whereas enhanced phosphorylation of Cas was observed Csk- cells, in which Src kinases are activated. These results suggest that Src kinases are responsible for the integrin-mediated tyrosine phosphorylation of Cas. FAK seems not to be necessary for phosphorylation of Cas, but when autophosphorylated, FAK may recruit Src family kinases to phosphorylate Cas. Cas was found to form complexes with Src homology 2 (SH2) domain-containing signaling molecules, such as the SH2/SH3 adapter protein Crk, following integrin-induced tyrosine phosphorylation. Guanine nucleotide exchange factors C3G and Sos were found in the Cas-Crk complex upon integrin ligand binding. These observations suggest that Cas serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion.     

Molecular basis for regulation of Src by the docking protein p130Cas

The docking protein p130Cas (Cas) becomes tyrosine-phosphorylated in its central substrate domain in response to extracellular stimuli such as integrin-mediated cell adhesion, and transmits signals through interactions with various intracellular signaling molecules such as the adaptor protein Crk. Src-family kinases (SFKs) bind a specific site in the carboxyl-terminal region of Cas and subsequently SFKs phosphorylate progressively the substrate domain in Cas. In this study crystallography, mutagenesis and binding assays were used to understand the molecular basis for Cas interactions with SFKs. Tyrosine phosphorylation regulates binding of Cas to SFKs, and the primary site for this phosphorylation, Y762, has been proposed. A phosphorylated peptide corresponding to Cas residues 759MEDpYDYVHL767 containing the key phosphotyrosine was crystallized in complex with the SH3-SH2 domain of the SFK Lck. The results provide the first structural data for this protein-protein interaction. The motif in Cas 762pYDYV binds to the SH2 domain in a mode that mimics high-affinity ligands, involving dual contacts of Y762 and V765 with conserved residues in SFK SH2 domains. In addition, Y764 is in position to make an electrostatic contact after phosphorylation with a conserved SFK arginine that mediates interactions with other high-affinity SH2 binders. These new molecular data suggest that Cas may regulate activity of Src as a competing ligand to displace intramolecular interactions that occur in SFKs (between the C-terminal tail and the SH2 domain) and restrain and down-regulate the kinase in an inactive form.     

Processive phosphorylation of p130Cas by Src depends on SH3-polyproline interactions

Many in vivo substrates of Src family tyrosine kinases possess sequences conforming to Src homology 2 and 3 (SH2 and SH3) domain-binding motifs. One such substrate is p130Cas, a protein that is hyperphosphorylated in v-Src transformed cells. Cas contains a substrate domain consisting of 15 potential tyrosine phosphorylation sites, C- and N-terminal polyproline regions fitting the consensus sequence for SH3 domain ligands, and a YDYV motif that binds the Src SH2 domain when phosphorylated. In an effort to understand the mechanisms of processive phosphorylation, we have explored the regions of Cas necessary for interaction with Src using the yeast two-hybrid system. Mutations in the SH2 domain-binding region of Cas or the Src SH2 domain have little effect in Cas-Src complex formation or phosphorylation. However, disruption of the C-terminal polyproline region of Cas completely abolishes interaction between the two proteins and results in impaired phosphorylation of Cas. Kinetic analyses using purified proteins indicated that multisite phosphorylation of Cas by Src follows a processive rather than a distributive mechanism. Furthermore, the kinetic studies show that there are two properties of the polyproline region of Cas that are important in enhancing substrate phosphorylation. First, the C-terminal polyproline serves to activate Src kinases through the process of SH3 domain displacement. Second, this region aids in anchoring the kinase to Cas to facilitate processive phosphorylation of the substrate domain. The two processes combine to ensure phosphorylation of Cas with high efficiency.     

Crystal structure of human macrophage elastase (MMP-12) in complex with a hydroxamic acid inhibitor

Human macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) that plays, like other members of the family, an important role in inflammatory processes contributing to tissue remodelling and destruction. In particular, a prominent role of MMP-12 in the destruction of elastin in the lung alveolar wall and the pathogenesis of emphysema has been suggested. It is therefore an attractive therapeutic target. We describe here the crystal structure of the catalytic domain of MMP-12 in complex with a hydroxamic acid inhibitor, CGS27023A. MMP-12 adopts the typical MMP fold and binds a structural zinc ion and three calcium ions in addition to the catalytic zinc ion. The enzyme structure shows an ordered N terminus close to the active site that is identical in conformation with the superactivated form of MMP-8. The S1'-specificity pocket is large and extends into a channel through the protein, which puts MMP-12 into the class of MMPs 3, 8 and 13 with large and open specificity pockets. The two crystallographically independent molecules adopt different conformations of the S1'-loop and its neighbouring loop due to differing crystal packing environments, suggesting that flexibility or the possibility of structural adjustments of these loop segments are intrinsic features of the MMP-12 structure and probably a common feature for all MMPs. The inhibitor binds in a bidentate fashion to the catalytic zinc ion. Its polar groups form hydrogen bonds in a substrate-like manner with beta-strand sIV of the enzyme, while the hydrophobic substituents are either positioned on the protein surface and are solvent-exposed or fill the upper part of the specificity pocket. The present structure enables us to aid the design of potent and selective inhibitors for MMP-12.     

A role for p130Cas in mechanotransduction

Focal adhesions are sites of contact between cells and the extracellular matrix. Sawada et al. (2006) now report that the mechanical stretching of cells forces p130Cas, an adaptor protein at focal adhesions, to undergo a conformational change. This change promotes phosphorylation of p130Cas by Src family kinases and the transduction of integrin-mediated signaling.     

A novel Cas family member, HEPL, regulates FAK and cell spreading

For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associated substrate) scaffolding protein family. Cas proteins mediate integrin-dependent signals at focal adhesions, regulating cell invasion and survival; at least one family member, HEF1, regulates mitosis. We here report a previously undescribed novel branch of the Cas protein family, designated HEPL (for HEF1-Efs-p130Cas-like). The HEPL branch is evolutionarily conserved through jawed vertebrates, and HEPL is found in some species lacking other members of the Cas family. The human HEPL mRNA and protein are selectively expressed in specific primary tissues and cancer cell lines, and HEPL maintains Cas family function in localization to focal adhesions, as well as regulation of FAK activity, focal adhesion integrity, and cell spreading. It has recently been demonstrated that upregulation of HEF1 expression marks and induces metastasis, whereas high endogenous levels of p130Cas are associated with poor prognosis in breast cancer, emphasizing the clinical relevance of Cas proteins. Better understanding of the complete protein family should help inform prediction of cancer incidence and prognosis.