Identification of Novel Crk-associated Substrate (p130Cas) Variants with Functionally Distinct Focal Adhesion Kinase Binding Activities

Elevated levels of p130^Cas (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1 gene) are associated with aggressiveness of breast tumors. Following phosphorylation of its substrate domain, p130^Cas promotes the integration of protein complexes involved in multiple signaling pathways and mediates cell proliferation, adhesion, and migration. In addition to the known BCAR1-1A (wild-type) and 1C variants, we identified four novel BCAR1 mRNA variants, generated by alternative first exon usage (1B, 1B1, 1D, and 1E). Exons 1A and 1C encode for four amino acids (aa), whereas 1D and 1E encode for 22 aa and 1B1 encodes for 50 aa. Exon 1B is non-coding, resulting in a truncated p130^Cas protein (Cas1B). BCAR1-1A, 1B1, and variant 1C mRNAs were ubiquitously expressed in cell lines and a survey of human tissues, whereas 1B, 1D, and 1E expression was more restricted. Reconstitution of all isoforms except for 1B in p130^Cas-deficient murine fibroblasts induced lamellipodia formation and membrane ruffling, which was unrelated to the substrate domain phosphorylation status. The longer isoforms exhibited increased binding to focal adhesion kinase (FAK), a molecule important for migration and adhesion. The shorter 1B isoform exhibited diminished FAK binding activity and significantly reduced migration and invasion. In contrast, the longest variant 1B1 established the most efficient FAK binding and greatly enhanced migration. Our results indicate that the p130^Cas exon 1 variants display altered functional properties. The truncated variant 1B and the longer isoform 1B1 may contribute to the diverse effects of p130^Cas on cell biology and therefore will be the target of future studies.     

黏着斑激酶与细胞迁移

细胞迁移过程始于细胞前端板状伪足的形成、外周黏附的建立、细胞体的收缩和尾部的解离.黏着斑激酶是一种非受体酪氨酸蛋白激酶,通过其激酶活性和"脚手架"的功能在细胞迁移的各个过程中发挥关键作用.现重点介绍黏着斑激酶介导的信号转导通路及其在调控细胞迁移方面的研究进展.     

The Lysyl Oxidase Pro-peptide Attenuates Fibronectin-mediated Activation of Focal Adhesion Kinase and p130Cas in Breast Cancer Cells

The lysyl oxidase (LOX) gene encodes an enzyme (LOX) critical for extracellular matrix maturation. The LOX gene has also been shown to inhibit the transforming activity of Ras oncogene signaling. In particular, the pro-peptide domain (LOX-PP) released from the secreted precursor protein (Pro-LOX) was found to inhibit the transformed phenotype of breast, lung, and pancreatic cancer cells. However, the mechanisms of action of LOX-PP remained to be determined. Here, the ability of LOX-PP to attenuate the integrin signaling pathway, which leads to phosphorylation of focal adhesion kinase (FAK), and the activation of its downstream target p130^Cas, was determined. In NF639 breast cancer cells driven by Her-2/neu, which signals via Ras, ectopic Pro-LOX and LOX-PP expression inhibited fibronectin-stimulated protein tyrosine phosphorylation. Importantly, phosphorylation of FAK on Tyr-397 and Tyr-576, and p130^Cas were substantially reduced. The amount of endogenous p130^Cas in the Triton X-100-insoluble protein fraction, and fibronectin-activated haptotaxis were decreased. Interestingly, expression of mature LOX enzyme enhanced fibronectin-stimulated integrin signaling. Of note, treatment with recombinant LOX-PP selectively reduced fibronectin-mediated haptotaxis of NF639, MDA-MB-231, and Hs578T breast cancer cells. Thus, evidence is provided that one mechanism of action of LOX-PP tumor suppression is to block fibronectin-stimulated signaling and cell migration.The lysyl oxidase (LOX)² gene family is comprised of five members LOX, LOXL1, LOXL2, LOXL3, and LOXL4, which encode enzymes that modify extracellular matrix (ECM) proteins to promote their cross-linking and deposition (1). The LOX gene is the best characterized and codes for the synthesis of a secreted 50-kDa glycosylated pro-enzyme (Pro-LOX). Pro-LOX is extracellularly processed by proteolytic cleavage to a mature active 32-kDa enzyme (LOX) and an 18-kDa pro-peptide (LOX-PP) by the procollagen C proteinases bone morphogenic protein-1 (BMP-1), and the related tolloid-like proteins TLL1 and TLL2 (2–4). In murine Pro-LOX, proteolytic processing occurs between amino acids Gly-162 and Asp-163, generating LOX-PP containing 141 amino acids (5). LOX-PP contains two consensus N-glycosylation sites, Asn-91 and Asn-138 (murine sequence) (2) and several O-glycosylation sites.³ LOX-PP does not contain any known protein domains, and structural prediction analysis indicates that LOX-PP assembles as an intrinsically disordered protein (6). Among the LOX family members, the C-terminal ends encode the enzyme domain and are highly conserved, whereas the N-terminal ends that encode the pro-peptide region have variable sequences. Based on structural and sequence similarities of the pro-peptide regions, the LOX family members can be divided into two subgroups: LOXL2, LOXL3, and LOXL4 as one group whose propeptide regions contain four scavenger receptor cysteine-rich domains, and LOX and LOXL1 as a separate group with much simpler and smaller pro-peptide region containing no cysteine residues (reviewed in Ref. 1). In contrast to Pro-LOX, the exact maturation site of Pro-LOXL1 is still unidentified.LOX is essential in the formation of blood vessels and in maintaining their normal characteristics (7–9). Up-regulation of LOX expression has been described in stromal cells that surround ductal breast and broncho-pulmonary carcinomas (10).Expression of the LOX gene was found to inhibit the transforming activity of the Ras oncogene in NIH 3T3 fibroblasts and hence was named the “ras recision” gene (rrg) (11, 12). The LOX gene was shown to inhibit growth in soft agar of NIH 3T3 fibroblasts and to attenuate Ras-mediated activation of phosphatidylinositol 3-kinase (PI3K), Akt, and Erk1/2 kinases and NF-κB activation (13). More recently, the rrg activity was mapped to the 18-kDa LOX-PP. Specifically, LOX-PP was shown to inhibit Ras-mediated transformation of fibroblasts as determined by reduced growth in soft agar, localization of PDK1 to the membrane, and activation of NF-κB (14). Furthermore, the inhibitory effects of LOX-PP on Ras signaling were extended to breast, pancreatic, and lung cancer cells (6, 14, 15). LOX-PP expression in these carcinoma cells reverted Her-2/neu- and Ras-mediated epithelial to mesenchymal transition (EMT), leading to increased expression of E-cadherin and γ-catenin, and reduced levels of Snail, vimentin, and/or BCL-2 (7, 15). Furthermore, LOX-PP expression reduced tumor formation in a xenograft model by Her-2/neu-overexpressing NF639 cells (6).Acquisition of the ability to invade the ECM is essential to EMT. The ECM has multiple mechanical and signaling functions. The ECM defines interfaces between tissues, provides a scaffold for cell traction, and a substrate for cell migration and adhesion. It is composed of a complex of proteins such as collagens, fibronectin, and laminin, which can interact and bind various growth factors (16). Fibronectin is of particular interest because it was recently shown to interact with the C terminus of Pro-LOX (17). Binding of fibronectin to its receptors (e.g. integrins α₅β₁ or α_vβ₁) stimulates the tyrosine phosphorylation of cellular proteins, in particular that of focal adhesion kinase (FAK) (18). Little is known about the mechanism of action of LOX-PP. Here, we have asked whether the tumor suppressor activity of LOX-PP attenuates the activation of the integrin signaling pathway in breast cancer cells. We report that LOX-PP attenuates FAK signaling and activation of its downstream target p130^Cas and is a robust inhibitor of fibronectin-stimulated cell migration.     

Tyrosine Y189 in the Substrate Domain of the Adhesion Docking Protein NEDD9 Is Conserved with p130Cas Y253 and Regulates NEDD9-Mediated Migration and Focal Adhesion Dynamics

The focal adhesion docking protein NEDD9/HEF1/Cas-L regulates cell migration and cancer invasion. NEDD9 is a member of the Cas family of proteins that share conserved overall protein-protein interaction domain structure, including a substrate domain that is characterized by extensive tyrosine (Y) phosphorylation. Previous studies have suggested that phosphorylation of Y253 in the substrate domain of the Cas family protein p130Cas is specifically required for p130Cas function in cell migration. While it is clear that tyrosine phosphorylation of the NEDD9 substrate domain is similarly required for the regulation of cell motility, whether individual NEDD9 tyrosine residues have discrete function in regulating motility has not previously been reported. In the present study we have used a global sequence alignment of Cas family proteins to identify a putative NEDD9 equivalent of p130Cas Y253. We find that NEDD9 Y189 aligns with p130Cas Y253 and that it is conserved among NEDD9 vertebrate orthologues. Expression of NEDD9 in which Y189 is mutated to phenylalanine results in increased rates of cell migration and is correlated with increased disassembly of GFP.NEDD9 focal adhesions. Conversely, mutation to Y189D significantly inhibits cell migration. Our previous data has suggested that NEDD9 stabilizes focal adhesions and the present data therefore suggests that phosphorylation of Y189 NEDD9 is required for this function. These findings indicate that the individual tyrosine residues of the NEDD9 substrate domain may serve discrete functional roles. Given the important role of this protein in promoting cancer invasion, greater understanding of the function of the individual tyrosine residues is important for the future design of approaches to target NEDD9 to arrest cancer cell invasion.     

JNK Pathway-associated Phosphatase Dephosphorylates Focal Adhesion Kinase and Suppresses Cell Migration

JNK pathway-associated phosphatase (JKAP, also named DUSP22) is expressed in various tissues, indicating that JKAP may have an important biological function. We showed that JKAP localized in the actin filament-enriched region. Expression of JKAP reduced cell migration, whereas a JKAP mutant lacking catalytic activity promoted cell motility. JKAP efficiently removed tyrosine phosphorylation of several proteins. We have identified focal adhesion kinase (FAK) as a substrate of JKAP. Overexpression of JKAP, but not JKAP mutant lacking catalytic activity, decreased FAK phosphorylation at tyrosines 397, 576, and 577 in H1299 cells. Consistent with these results, decreasing JKAP expression by RNA interference promoted cell migration and Src-induced FAK phosphorylation. Taken together, this study identified a new role for JKAP in the modulation of FAK phosphorylation and cell motility.     

Cleavage of p130Cas in anoikis

p130Cas is a multifunctional signaling adaptor protein. It integrates and relays signals generated from a variety of extracellular stimuli and regulates a number of cellular activities including cell death. In this study, we analyzed the regulation and function of p130Cas in anoikis, a type of apoptosis caused by disruption of cell-matrix interactions. We found that p130Cas was specifically cleaved during anoikis in anoikis-sensitive epithelial cells, but not in anoikis-resistant tumor cells. There is a close correlation between p130Cas cleavage and anoikis. Furthermore, we found that the cleavage of p130Cas, as well as another focal adhesion component FAK, is different from that of caspase substrate PARP and spectrin. Although caspases and calpain were found to be involved in the cleavage of p130Cas, there appear to be other unidentified proteases that are mainly responsible for the cleavage of p130Cas, particularly at the early stage of anoikis. Overexpression of the p130Cas cleavage product induced apoptosis. Taken together, these data suggest that there are novel proteases involved in the cleavage of p130Cas during anoikis, which may be functionally involved in the onset of anoikis. p130Cas may have a dual role in the regulation of anoikis. On one hand, it mediates a survival signal from cell-matrix interactions when cells are attached to the extracellular matrix. On the other hand, it participates in executing cell death when cell-matrix interactions are disrupted. These observations provide new insights into the understanding of the function of p130Cas and the molecular mechanism of anoikis.     

Quantitative Proteomic Profiling Identifies DPYSL3 as Pancreatic Ductal Adenocarcinoma-Associated Molecule That Regulates Cell Adhesion and Migration by Stabilization of Focal Adhesion Complex

Elucidation of how pancreatic cancer cells give rise to distant metastasis is urgently needed in order to provide not only a better understanding of the underlying molecular mechanisms, but also to identify novel targets for greatly improved molecular diagnosis and therapeutic intervention. We employed combined proteomic technologies including mass spectrometry and isobaric tags for relative and absolute quantification peptide tagging to analyze protein profiles of surgically resected human pancreatic ductal adenocarcinoma tissues. We identified a protein, dihydropyrimidinase-like 3, as highly expressed in human pancreatic ductal adenocarcinoma tissues as well as pancreatic cancer cell lines. Characterization of the roles of dihydropyrimidinase-like 3 in relation to cancer cell adhesion and migration in vitro, and metastasis in vivo was performed using a series of functional analyses, including those employing multiple reaction monitoring proteomic analysis. Furthermore, dihydropyrimidinase-like 3 was found to interact with Ezrin, which has important roles in cell adhesion, motility, and invasion, while that interaction promoted stabilization of an adhesion complex consisting of Ezrin, c-Src, focal adhesion kinase, and Talin1. We also found that exogenous expression of dihydropyrimidinase-like 3 induced activating phosphorylation of Ezrin and c-Src, leading to up-regulation of the signaling pathway. Taken together, the present results indicate successful application of combined proteomic approaches to identify a novel key player, dihydropyrimidinase-like 3, in pancreatic ductal adenocarcinoma tumorigenesis, which may serve as an important biomarker and/or drug target to improve therapeutic strategies.     

Structural Evolution of the Protein Kinase–Like Superfamily

The protein kinase family is large and important, but it is only one family in a larger superfamily of homologous kinases that phosphorylate a variety of substrates and play important roles in all three superkingdoms of life. We used a carefully constructed structural alignment of selected kinases as the basis for a study of the structural evolution of the protein kinase–like superfamily. The comparison of structures revealed a “universal core” domain consisting only of regions required for ATP binding and the phosphotransfer reaction. Remarkably, even within the universal core some kinase structures display notable changes, while still retaining essential activity. Hence, the protein kinase–like superfamily has undergone substantial structural and sequence revision over long evolutionary timescales. We constructed a phylogenetic tree for the superfamily using a novel approach that allowed for the combination of sequence and structure information into a unified quantitative analysis. When considered against the backdrop of species distribution and other metrics, our tree provides a compelling scenario for the development of the various kinase families from a shared common ancestor. We propose that most of the so-called “atypical kinases” are not intermittently derived from protein kinases, but rather diverged early in evolution to form a distinct phyletic group. Within the atypical kinases, the aminoglycoside and choline kinase families appear to share the closest relationship. These two families in turn appear to be the most closely related to the protein kinase family. In addition, our analysis suggests that the actin-fragmin kinase, an atypical protein kinase, is more closely related to the phosphoinositide-3 kinase family than to the protein kinase family. The two most divergent families, α-kinases and phosphatidylinositol phosphate kinases (PIPKs), appear to have distinct evolutionary histories. While the PIPKs probably have an evolutionary relationship with the rest of the kinase superfamily, the relationship appears to be very distant (and perhaps indirect). Conversely, the α-kinases appear to be an exception to the scenario of early divergence for the atypical kinases: they apparently arose relatively recently in eukaryotes. We present possible scenarios for the derivation of the α-kinases from an extant kinase fold.     

Identification of two focal adhesion targeting sequences in the adapter molecule p130(Cas)

The adapter molecule CAS is localized primarily within focal adhesions in fibroblasts. Because many of the cellular functions attributed to CAS are likely to be dependent on its presence in focal adhesions, this study was undertaken to identify regions of the protein that are involved in its localization. The SH3 domain of CAS, when expressed in isolation from the rest of the protein, was able to target to focal adhesions, whereas a variant containing a point mutation that rendered the SH3 domain unable to associate with FAK remained cytoplasmic. However, in the context of full-length CAS, this mutation did not prevent CAS localization to focal adhesions. Two other variants of CAS that contained deletions of either the SH3 domain alone, or the SH3 domain together with an adjoining proline-rich region, also retained the capacity to localize to focal adhesions. A second focal adhesion targeting region was mapped to the extreme carboxy terminus of CAS. The identification of this second focal adhesion targeting domain in CAS ascribes a previously unknown function to the highly conserved C terminus of CAS. The regulated targeting of CAS to focal adhesions by two independent domains may reflect the important role of CAS within this subcellular compartment.     

Identification of Trans-Sialidases as a Common Mediator of Endothelial Cell Activation by African Trypanosomes

Understanding African Trypanosomiasis (AT) host-pathogen interaction is the key to an “anti-disease vaccine”, a novel strategy to control AT. Here we provide a better insight into this poorly described interaction by characterizing the activation of a panel of endothelial cells by bloodstream forms of four African trypanosome species, known to interact with host endothelium. T. congolense, T. vivax, and T. b. gambiense activated the endothelial NF-κB pathway, but interestingly, not T. b. brucei. The parasitic TS (trans-sialidases) mediated this NF-κB activation, remarkably via their lectin-like domain and induced production of pro-inflammatory molecules not only in vitro but also in vivo, suggesting a considerable impact on pathogenesis. For the first time, TS activity was identified in T. b. gambiense BSF which distinguishes it from the subspecies T. b. brucei. The corresponding TS were characterized and shown to activate endothelial cells, suggesting that TS represent a common mediator of endothelium activation among trypanosome species with divergent physiopathologies.     

BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling

The nonreceptor protein-tyrosine kinase c-Src is frequently overexpressed and/or activated in a variety of cancers, including those of the breast. Several heterologous binding partners of c-Src have been shown to regulate its catalytic activity by relieving intramolecular autoinhibitory interactions. One such protein, p130^Cas (Cas), is expressed at high levels in both breast cancer cell lines and breast tumors, providing a potential mechanism for c-Src activation in breast cancers. The Cas-binding protein BCAR3 (breast cancer antiestrogen resistance-3) is expressed at high levels in invasive breast cancer cell lines, and this molecule has previously been shown to coordinate with Cas to increase c-Src activity in COS-1 cells. In this study, we show for the first time using gain- and loss-of-function approaches that BCAR3 regulates c-Src activity in the endogenous setting of breast cancer cells. We further show that BCAR3 regulates the interaction between Cas and c-Src, both qualitatively as well as quantitatively. Finally, we present evidence that the coordinated activity of these proteins contributes to breast cancer cell adhesion signaling and spreading. Based on these data, we propose that the c-Src/Cas/BCAR3 signaling axis is a prominent regulator of c-Src activity, which in turn controls cell behaviors that lead to aggressive and invasive breast tumor phenotypes.