Structural Analysis of the Interactions Between Paxillin LD Motifs and α-Parvin

Lorenz, Sonja; Vakonakis, Ioannis; Lowe, E.D.; Campbell, Iain D.; Noble, M.E.M.; Hoellerer, M.K.

doi:10.1016/j.str.2008.08.007

Cited by 34 publications

(62 citation statements)

References 44 publications

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“…Association with paxillin is required for actopaxin and integrin-linked kinase targeting to focal adhesions (29) and promotes adhesion formation (27). Recent structural studies indicate that separation of the LD1 domain from paxillin would alter interactions with key players such as actopaxin and integrin-linked kinase and may modulate adhesion dynamics (30,31). In accordance, we observed altered adhesion dynamics upon expression of paxillin delta and the paxillin calpain cleavage fragment, which lack the LD1 domain (Fig.…”

Section: Discussionsupporting

confidence: 83%

Calpain-mediated Proteolysis of Paxillin Negatively Regulates Focal Adhesion Dynamics and Cell Migration

Cortesio

Boateng²,

Piazza³

et al. 2011

Journal of Biological Chemistry

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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...

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Section: Discussionsupporting

confidence: 83%

Calpain-mediated Proteolysis of Paxillin Negatively Regulates Focal Adhesion Dynamics and Cell Migration

Cortesio

Boateng²,

Piazza³

et al. 2011

Journal of Biological Chemistry

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“…In addition to being required for SLK-mediated phosphorylation, the altered conformation of paxillin resulting from the mutation of serines 243 and 244 to glycines would suggest that LD3 is important in maintaining the integrity of the tertiary structure of the protein. Although the crystal structures of paxillin motifs LD2 and LD4 have been resolved, the crystal structure of the entire protein has yet to be determined [103,158,159]. Consequently, it is difficult to draw any definitive conclusions about the paxillin structure from these results alone.…”

Section: Discussionmentioning

confidence: 99%

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

Baron

et al. 2012

Oncogene

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“…The PDB accession code for FAK is 1OW6 (31) and for Pyk2 is 3GM1 (32). ality was ascribed to the "pseudo-palindromic character of the LD consensus" (45). There are other examples of directionality being imposed on protein binding to highly similar peptides; for example, the binding direction of PXXP motifs to SH3 domains is determined by the presence of an arginine residue N or C terminus to the PXXP motif (46).…”

Section: Directionality Of Ld Motifmentioning

confidence: 99%

Molecular Recognition of Leucine-Aspartate Repeat (LD) Motifs by the Focal Adhesion Targeting Homology Domain of Cerebral Cavernous Malformation 3 (CCM3)

Zhang

et al. 2011

Journal of Biological Chemistry

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Cerebral cavernous malformation (CCM) is a disease that affects between 0.1 and 0.5% of the human population, with mutations in CCM3 accounting for ϳ15% of the autosomal dominant form of the disease. We recently reported that CCM3 contains an N-terminal dimerization domain (CCM3D) and a C-terminal focal adhesion targeting (FAT) homology domain. Intermolecular protein-protein interactions of CCM3 are mediated by a highly conserved surface on the FAT homology domain and are affected by CCM3 truncations in the human disease. Here we report the crystal structures of CCM3 in complex with three different leucine-aspartate repeat (LD) motifs (LD1, LD2, and LD4) from the scaffolding protein paxillin, at 2.8, 2.7, and 2.5 Å resolution. We show that CCM3 binds LD motifs using the highly conserved hydrophobic patch 1 (HP1) and that this binding is similar to the binding of focal adhesion kinase and Pyk2 FAT domains to paxillin LD motifs. We further show by surface plasmon resonance that CCM3 binds paxillin LD motifs with affinities in the micromolar range, similar to FAK family FAT domains. Finally, we show that endogenous CCM3 and paxillin co-localize in mouse cerebral pericytes. These studies provide a molecular-level framework to investigate the protein-protein interactions of CCM3.Mutations in three genes, CCM1, CCM2, and CCM3, are implicated in the inherited autosomal dominant form of cerebral cavernous malformation (CCM) 2 (1, 2). CCM is a dysplasia that affects the central nervous system, resulting in thin-walled, dilated blood vessels. Complications resulting from CCM include hemorrhagic stroke, seizure, epilepsy, and other focal neurological outcomes (3)(4)(5). Approximately 15% of inherited CCM cases are associated with truncations or mutations in CCM3, the protein product of which is cerebral cavernous malformation 3 (CCM3, also called PDCD10 (programmed cell death 10), or TFAR15 (TF-1 cell apoptosis-related protein 15) (6, 7). The function of this protein is not fully described, and its protein binding partners are still being determined; however, both global and endothelial-specific deletions of CCM3 are incompatible with life (8). This protein seems to have a scaffolding function and has been shown to directly interact with CCM2 (9 -11), germinal center kinase III family members STK24, STK25, and MST4 (10, 12-17), the STRIPAK complex (18), and paxillin (11) and can associate the cytoplasmic tail of VEGFR2 (8). The atomic-level mechanisms of CCM3 interactions with its binding partners have not been described.Paxillin is a scaffolding protein important for localization of many proteins to the intracellular side of cell adhesions (19 -21). Importantly, the localization of protein kinases, phosphatases, and GTPase regulating proteins (guanine nucleotide exchange factors, GTPase-activating proteins, and effectors) to the actin cytoskeleton and the spatial-temporal dynamics of cellular adhesion are impacted by paxillin (22-24). Examples of proteins that paxillin recruits include FAK, Pyk2, PTP-PEST, CrkII, DOCK180,...

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Structural Analysis of the Interactions Between Paxillin LD Motifs and α-Parvin

Cited by 34 publications

References 44 publications

Calpain-mediated Proteolysis of Paxillin Negatively Regulates Focal Adhesion Dynamics and Cell Migration

Calpain-mediated Proteolysis of Paxillin Negatively Regulates Focal Adhesion Dynamics and Cell Migration

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

Molecular Recognition of Leucine-Aspartate Repeat (LD) Motifs by the Focal Adhesion Targeting Homology Domain of Cerebral Cavernous Malformation 3 (CCM3)

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