The dynamic turnover of integrin-mediated adhesions is important for cell migration. Paxillin is an adaptor protein that localizes to focal adhesions and has been implicated in cell motility. We previously reported that calpain-mediated proteolysis of talin1 and focal adhesion kinase mediates adhesion disassembly in motile cells. To determine whether calpain-mediated paxillin proteolysis regulates focal adhesion dynamics and cell motility, we mapped the preferred calpain proteolytic site in paxillin. The cleavage site is between the paxillin LD1 and LD2 motifs and generates a C-terminal fragment that is similar in size to the alternative product paxillin delta. The calpain-generated proteolytic fragment, like paxillin delta, functions as a paxillin antagonist and impairs focal adhesion disassembly and migration. We generated mutant paxillin with a point mutation (S95G) that renders it partially resistant to calpain proteolysis. Paxillin-deficient cells that express paxillin S95G display increased turnover of zyxin-containing adhesions using timelapse microscopy and also show increased migration. Moreover, cancer-associated somatic mutations in paxillin are common in the N-terminal region between the LD1 and LD2 motifs and confer partial calpain resistance. Taken together, these findings suggest a novel role for calpain-mediated proteolysis of paxillin as a negative regulator of focal adhesion dynamics and migration that may function to limit cancer cell invasion.The dynamic regulation of integrin-mediated adhesions is important for cell migration in both normal and pathological processes, including tumor invasion and metastasis. Integrinmediated adhesions assemble at the leading edge of cells and establish a structural link between the extracellular matrix and the actin cytoskeleton. The correct spatial and temporal control of adhesion formation and disassembly at both the leading and trailing edge of cells is important for efficient cell migration. However, the mechanisms that regulate the turnover of adhesions in motile cells are still not well understood.Paxillin is an adaptor and phospho-protein that localizes to focal adhesions. Paxillin serves an important scaffolding function at focal adhesions by recruiting and binding to many signaling and structural proteins (1). First identified as a 68-kDa protein that was heavily phosphorylated in Src-transformed fibroblasts (2), Src-mediated phosphorylation of paxillin at Tyr-31 and Tyr-118 is important for focal adhesion disassembly at the leading edge of migrating cells (3). Paxillin expression and function is highly regulated by both alternative splicing and posttranslational modifications (4). An internal, alternative translation start site in paxillin is conserved in all species and produces the paxillin isoform known as paxillin delta (4), which functions as a paxillin antagonist that impairs cell migration (5).Paxillin can also be modified by calpain-mediated proteolysis (6, 7). Calpains are a family of calcium-dependent, intracellular cysteine proteases tha...

No abstract

There were errors published in J. Cell Sci. 125, 1329-1341.All mentions of Tyr340 and Tyr350 should read Tyr337 and Tyr347, respectively.In addition, the double phospho-mutant should be mCherry-mAbp1(Y337E,Y347F).The corrected legend to Fig. 9 appears below. Fig. 9. Model of mAbp1 regulation of podosome formation and invasive migration. mAbp1 binds to filamentous actin and is phosphorylated by Src at Y337 and Y347 in the proline-rich region (oval). Phosphorylation of mAbp1 at Y347 is required for podosome rosette formation and the inhibition of invasive migration. Y347 might be the crucial residue that mediates maturation of podosome dots to rosettes. Conversely, Src phosphorylation of both Y337 and Y347 impairs formation of podosome dots.We apologise for these errors. Summary Podosomes are dynamic actin-based structures that mediate adhesion to the extracellular matrix and localize matrix degradation to facilitate cell motility and invasion. Drebrin-like protein (DBNL), which is homologous to yeast mAbp1 and is therefore known as mammalian actin-binding protein 1 (mAbp1), has been implicated in receptor-mediated endocytosis, vesicle recycling and dorsal ruffle formation. However, it is not known whether mAbp1 regulates podosome formation or cell invasion. In this study, we found that mAbp1 localizes to podosomes and is necessary for the formation of podosome rosettes in Src-transformed fibroblasts. Despite their structural similarity, mAbp1 and cortactin play distinct roles in podosome regulation. Cortactin was necessary for the formation of podosome dots, whereas mAbp1 was necessary for the formation of organized podosome rosettes in Src-transformed cells. We identified specific Src phosphorylation sites, Tyr337 and Tyr347 of mAbp1, which mediate the formation of podosome rosettes and degradation of the ECM. In contrast to dorsal ruffles, the interaction of mAbp1 with WASP-interacting protein (WIP) was not necessary for the formation of podosome rosettes. Finally, we showed that depletion of mAbp1 increased invasive cell migration, suggesting that mAbp1 differentially regulates matrix degradation and cell invasion. Collectively, our findings identify a role for mAbp1 in podosome rosette formation and cell invasion downstream of Src.

Mammalian actin-binding protein-1 (mAbp1) is an adaptor protein that binds actin and modulates scission during endocytosis. Recent studies suggest that mAbp1 impairs cell invasion; however, the mechanism for the inhibitory effects of mAbp1 remain unclear. We performed a yeast two-hybrid screen and identified the adaptor protein, FHL2, as a novel binding partner that interacts with the N-terminal actin depolymerizing factor homology domain (ADFH) domain of mAbp1. Here we report that depletion of mAbp1 or ectopic expression of the ADFH domain of mAbp1 increased Rho GTPase signaling and breast cancer cell invasion. Moreover, cell invasion induced by the ADFH domain of mAbp1 required the expression of FHL2. Taken together, our findings show that mAbp1 and FHL2 are novel binding partners that differentially regulate Rho GTPase signaling and MTLn3 breast cancer cell invasion.Invasive migration of cancer cells is dependent on a dynamic actin cytoskeleton (1). Key regulators of the actin cytoskeleton include actin-binding proteins like cortactin, which have been implicated in invadopodia and podosome formation and invasive cell migration. Mammalian actin-binding protein-1 (mAbp1, 2 Hip-55, DBNL) has significant structural homology to cortactin and binds actin with an N-terminal ADFH domain (2). Both mAbp1 and cortactin have identified roles during endocytosis and vesicle trafficking (3-5), and both proteins localize to lamellipodia, podosomes, and dorsal ruffles (4, 6, 7). However, in contrast to cortactin, mAbp1 impairs podosome formation and invasive migration of Src-transformed fibroblasts (8).To determine how mAbp1 regulates cell invasion, we performed a yeast two-hybrid screen and identified the fourand-a-half LIM domain protein 2 (FHL2) as a novel mAbp1-binding partner that does not interact with cortactin. FHL2 (also known as DRAL or SLIM3) is a LIM domain protein consisting of four-and-a-half LIM domains. Each LIM domain contains two zinc finger loops that mediate proteinprotein interactions. FHL2 interacts with Ͼ50 proteins (9) including integrins and focal adhesion kinase (FAK) (10, 11), actin (12), and the transcription factors estrogen receptor (ER), activator protein-1 (AP-1), and ␤-catenin (13-18). Interestingly, FHL2 is expressed at low levels in many tissues like breast, placenta, uterus, and the lungs (17, 19) and is up-regulated in cancer. For example, FHL2 expression is increased in many cancers including breast (20, 21), ovarian (22), prostate (23), lung (24), colon (25), brain (26), and skin cancers (27). In primary breast tumors, patients with high levels of FHL2 have a poorer survival rate (21), suggesting that FHL2 may be involved in cancer progression. Furthermore, overexpression of FHL2 in MDA-MB-231 human breast cancer cells enhances colony formation in soft agar, whereas knockdown reduces the ability to form colonies (20).Here we report a novel interaction between the N-terminal ADFH domain of mAbp1 and FHL2 that modulates cell invasion. In the yeast, but not the mammalian form, the N terminu...

No abstract