FAK and paxillin: regulators of N-cadherin adhesion and inhibitors of cell migration?

FAK and paxillin are important components in integrin-regulated signaling. New evidence suggests that these two proteins function in crosstalk between cell–matrix and cell–cell adhesions. Further, new insight suggests that under some conditions these proteins inhibit cell migration, in contrast to their established roles in several cell systems as positive regulators of cell adhesion and migration

Phosphorylation of paxillin by p38MAPK is involved in the neurite extension of PC-12 cells

Cell adhesions play an important role in neurite extension. Paxillin, a focal adhesion adaptor protein involved in focal adhesion dynamics, has been demonstrated to be required for neurite outgrowth. However, the molecular mechanism by which paxillin regulates neurite outgrowth is unknown. Here, we show that paxillin is phosphorylated by p38MAPK in vitro and in nerve growth factor (NGF)–induced PC-12 cells. Ser 85 (Ser 83 for endogenous paxillin) is identified as one of major phosphorylation sites by phosphopeptide mapping and mass spectrometry. Moreover, expression of the Ser 85 → Ala mutant of paxillin (paxS85A) significantly inhibits NGF-induced neurite extension of PC-12 cells, whereas expression of wild-type (wt) paxillin does not influence neurite outgrowth. Further experiments indicate that cells expressing paxS85A exhibit small, clustered focal adhesions which are not normally seen in cells expressing wt paxillin. Although wt paxillin and paxS85A have the same ability to bind vinculin and focal adhesion kinase, wt paxillin more efficiently associates with Pyk2 than paxS85A. Thus, phosphorylation of paxillin is involved in NGF-induced neurite extension of PC-12 cells, probably through regulating focal adhesion organization

Structural Basis for the Autoinhibition of Focal Adhesion Kinase

SummaryAppropriate tyrosine kinase signaling depends on coordinated sequential coupling of protein-protein interactions with catalytic activation. Focal adhesion kinase (FAK) integrates signals from integrin and growth factor receptors to regulate cellular responses including cell adhesion, migration, and survival. Here, we describe crystal structures representing both autoinhibited and active states of FAK. The inactive structure reveals a mechanism of inhibition in which the N-terminal FERM domain directly binds the kinase domain, blocking access to the catalytic cleft and protecting the FAK activation loop from Src phosphorylation. Additionally, the FERM domain sequesters the Tyr397 autophosphorylation and Src recruitment site, which lies in the linker connecting the FERM and kinase domains. The active phosphorylated FAK kinase adopts a conformation that is immune to FERM inhibition. Our biochemical and structural analysis shows how the architecture of autoinhibited FAK orchestrates an activation sequence of FERM domain displacement, linker autophosphorylation, Src recruitment, and full catalytic activation

The N Termini of Focal Adhesion Kinase Family Members Regulate Substrate Phosphorylation, Localization, and Cell Morphology

The focal adhesion kinase (FAK) and cell adhesion kinase beta (CAKbeta, PYK2, CADTK, RAFTK) are highly homologous FAK family members, yet clearly have unique roles in the cell. Comparative analyses of FAK and CAKbeta have revealed intriguing differences in their activities. These differences were investigated further through the characterization of a set of FAK/CAKbeta chimeric kinases. CAKbeta exhibited greater catalytic activity than FAK in vitro, providing a molecular basis for differential substrate phosphorylation by FAK and CAKbeta in vivo. Furthermore, the N terminus may regulate catalytic activity since chimeras containing the FAK N terminus and CAKbeta catalytic domain exhibited a striking high level of catalytic activity and substrate phosphorylation. Unexpectedly, a modulatory role for the N termini in subcellular localization was also revealed. Chimeras containing the FAK N terminus and CAKbeta C terminus localized to focal adhesions, whereas chimeras containing the N and C termini of CAKbeta did not. Finally, prominent changes in cell morphology were induced upon expression of chimeras containing the CAKbeta N terminus, which were not associated with apoptotic cell death, cell cycle progression delay, or changes in Rho activity. These results demonstrate novel regulatory roles for the N terminus of FAK family kinases

Multiple stimuli induce tyrosine phosphorylation of the Crk-binding sites of paxillin

Paxillin is a focal-adhesion-associated, tyrosine-phosphorylated protein. In cells transformed by the src, crk or BCR-Abl oncogenes, the phosphotyrosine content of paxillin is elevated. In normal cells paxillin functions in signalling following integrin-dependent cell adhesion or exposure to a number of stimuli, including growth factors and neuropeptides. These stimuli induce tyrosine phosphorylation of paxillin, regulating the association of Src homology 2 domain-containing signalling molecules with paxillin. There are multiple sites of tyrosine phosphorylation on paxillin. To elucidate the role of paxillin in transducing signals in response to various stimuli, it is essential to identify all of the sites of phosphorylation on paxillin and to define which residues are phosphorylated in response to distinct stimuli. We describe two new sites of tyrosine phosphorylation on paxillin, residues at positions 40 and 88. Using paxillin variants with phenylalanine substitutions at phosphorylation sites and phospho-specific paxillin antibodies, tyrosine phosphorylation of paxillin in response to distinct stimuli was examined. The results demonstrate that Tyr(31) and Tyr(118), which are binding sites for Crk, are major sites of tyrosine phosphorylation following cell adhesion or stimulation with platelet-derived growth factor or angiotensin II. Thus multiple stimuli may elicit similar signalling events downstream of paxillin

On Iron Enrichment, Star Formation, and Type Ia Supernovae in Galaxy Clusters

The nature of star formation and Type Ia supernovae (SNIa) in galaxies in the field and in rich galaxy clusters are contrasted by juxtaposing the build-up of heavy metals in the universe inferred from observed star formation and supernovae rate histories with data on the evolution of Fe abundances in the intracluster medium (ICM). Models for the chemical evolution of Fe in these environments are constructed, subject to observational constraints, for this purpose. While models with a mean delay for SNIa of 3 Gyr and standard initial mass function (IMF) are consistent with observations in the field, cluster Fe enrichment immediately tracks a rapid, top-heavy phase of star formation -- although transport of Fe into the ICM may be more prolonged and star formation likely continues to redshifts <1. The source of this prompt enrichment is Type II supernovae (SNII) yielding at least 0.1 solar masses per explosion (if the SNIa rate normalization is scaled down from its value in the field according to the relative number of candidate progenitor stars in the 3-8 solar mass range) and/or SNIa explosions with short delay times associated with the rapid star formation mode. Star formation is >3 times more efficient in rich clusters than in the field, mitigating the overcooling problem in numerical cluster simulations. Both the fraction of baryons cycled through stars, and the fraction of the total present-day stellar mass in the form of stellar remnants, are substantially greater in clusters than in the field.Comment: 51 pages including 26 figures and 2 tables, accepted for publication in ApJ 5/4/0

MAP kinases and cell migration

Recent studies have demonstrated that mitogen-activated protein kinases (MAPKs), including Jun N-terminus kinase (JNK), p38 and Erk, play crucial roles in cell migration. JNK, for example, regulates cell migration by phosphorylating paxillin, DCX, Jun and microtubule-associated proteins. Studies of p38 show that this MAPK modulates migration by phosphorylating MAPK-activated protein kinase 2/3 (MAPKAP 2/3), which appears to be important for directionality of migration. Erk governs cell movement by phosphorylating myosin light chain kinase (MLCK), calpain or FAK. Thus, the different kinases in the MAPK family all seem able to regulate cell migration but by distinct mechanisms

Multiple paxillin binding sites regulate FAK function

Abstract Background FAK localization to focal adhesions is essential for its activation and function. Localization of FAK is mediated through the C-terminal focal adhesion targeting (FAT) domain. Recent structural analyses have revealed two paxillin-binding sites in the FAT domain of FAK. To define the role of paxillin binding to each site on FAK, point mutations have been engineered to specifically disrupt paxillin binding to each docking site on the FAT domain of FAK individually or in combination. Results These mutants have been characterized and reveal an important role for paxillin binding in FAK subcellular localization and signaling. One paxillin-binding site (comprised of α-helices 1 and 4 of the FAT domain) plays a more prominent role in localization than the other. Mutation of either paxillin-binding site has similar effects on FAK activation and downstream signaling. However, the sites aren't strictly redundant as each mutant exhibits phosphorylation/signaling defects distinct from wild type FAK and a mutant completely defective for paxillin binding. Conclusion The studies demonstrate that the two paxillin-binding sites of FAK are not redundant and that both sites are required for FAK function

Discovery of an Unbound Hyper-Velocity Star in the Milky Way Halo

We have discovered a star, SDSS J090745.0+024507, leaving the Galaxy with a heliocentric radial velocity of +853+-12 km/s, the largest velocity ever observed in the Milky Way halo. The star is either a hot blue horizontal branch star or a B9 main sequence star with a heliocentric distance ~55 kpc. Corrected for the solar reflex motion and to the local standard of rest, the Galactic rest-frame velocity is +709 km/s. Because its radial velocity vector points 173.8 deg from the Galactic center, we suggest that this star is the first example of a hyper-velocity star ejected from the Galactic center as predicted by Hills and later discussed by Yu & Tremaine. The star has [Fe/H]~0, consistent with a Galactic center origin, and a travel time of <80 Myr from the Galactic center, consistent with its stellar lifetime. If the star is indeed traveling from the Galactic center, it should have a proper motion of 0.3 mas/yr observable with GAIA. Identifying additional hyper-velocity stars throughout the halo will constrain the production rate history of hyper-velocity stars at the Galactic center.Comment: 4 pages, submitted to ApJ Letter

Hypervelocity Stars: Predicting the Spectrum of Ejection Velocities

The disruption of binary stars by the tidal field of the black hole in the Galactic Center can produce the hypervelocity stars observed in the halo. We use numerical models to simulate the full spectrum of observable velocities of stars ejected into the halo by this binary disruption process. Our model includes a range of parameters for binaries with 3-4 M_Solar primaries, consideration of radial orbits of the ejected stars through an approximate mass distribution for the Galaxy, and the impact of stellar lifetimes. We calculate the spectrum of ejection velocities and reproduce previous results for the mean ejection velocity at the Galactic center. The model predicts that the full population of ejected stars includes both the hypervelocity stars with velocities large enough to escape from the Galaxy and a comparable number of ejected, but bound, stars of the same stellar type. The predicted median speeds of the population of ejected stars as a function of distance in the halo are consistent with current observations. Combining the model with the data also shows that interesting constraints on the properties of binaries in the Galactic Center and on the mass distribution in the Galaxy can be obtained even with modest samples of ejected stars.Comment: 26 pages, including 6 figures, accepted for publication in the Astrophysical Journa