No abstract

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton. In order to understand how Pyk2 signaling mediates these diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 that contains potential protein-protein interaction sites as bait. A prominent binder of Pyk2 identified by this method was the Arf-GTPase-activating protein ASAP1. Pyk2-ASAP1 interaction was confirmed in pull-down as well as in co-immunoprecipitation experiments, and contact sites were mapped to the proline-rich regions of Pyk2 and the SH3 domain of ASAP1. Pyk2 directly phosphorylates ASAP1 on tyrosine residues in vitro and increases ASAP1 tyrosine phosphorylation when co-expressed in HEK293T cells. Phosphorylation of tyrosine 308 and 782 affects the phosphoinositide binding profile of ASAP1, and fluorimetric Arf-GTPase assays with purified proteins revealed an inhibition of ASAP1 GTPase-activating protein activity by Pyk2-mediated tyrosine phosphorylation. We therefore provide evidence for a functional interaction between Pyk2 and ASAP1 and a regulation of ASAP1 and hence Arf1 activity by Pyk2-mediated tyrosine phosphorylation. Pyk2 and FAK1 comprise a distinct family of non-receptor protein tyrosine kinases that are involved in transmission of extracellular signals to intracellular kinase cascades and regulation of diverse cellular responses such as adhesion, proliferation, differentiation, and apoptosis (1). Pyk2 and FAK share about 45% amino acid identity and a similar domain structure: a unique N terminus containing a FERM (band 4

Prostaglandin D 2 activation of the seven-transmembrane receptor CRTH2 regulates numerous cell functions that are important in inflammatory diseases, such as asthma. Despite its disease implication, no studies to date aimed at identifying receptor domains governing signaling and surface expression of human CRTH2. We tested the hypothesis that CRTH2 may take advantage of its C-tail to silence its own signaling and that this mechanism may explain the poor functional responses observed with CRTH2 in heterologous expression systems. Although the C terminus is a critical determinant for retention of CRTH2 at the plasma membrane, the presence of this domain confers a signaling-compromised conformation onto the receptor. Indeed, a mutant receptor lacking the major portion of its C-terminal tail displays paradoxically enhanced G␣ i and ERK1/2 activation despite enhanced constitutive and agonist-mediated internalization. Enhanced activation of G␣ i proteins and downstream signaling cascades is probably due to the inability of the tail-truncated receptor to recruit ␤-arrestin2 and undergo homologous desensitization. Unexpectedly, CRTH2 is not phosphorylated upon agonist-stimulation, a primary mechanism by which GPCR activity is regulated. Dynamic mass redistribution assays, which allow label-free monitoring of all major G protein pathways in real time, confirm that the C terminus inhibits G␣ i signaling of CRTH2 but does not encode G protein specificity determinants. We propose that intrinsic CRTH2 inhibition by its C terminus may represent a rather unappreciated strategy employed by a GPCR to specify the extent of G protein activation and that this mechanism may compensate for the absence of the classical phosphorylation-dependent signal attenuation.2 is a lipid mediator that has been considered essential in the development of inflammatory diseases such as asthma and atopic dermatitis (1-3). It is the major cyclooxygenase metabolite synthesized in allergen-activated mast cells and is released upon their immunological activation (4). The biological effects of PGD 2 are mediated by two G protein-coupled receptors, DP1 and DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on T helper type 2 cells), respectively (5, 6). DP1 activation leads to G␣ s -mediated elevation of intracellular cyclic AMP, whereas activation of CRTH2 results in an increase in intracellular Ca 2ϩ levels via the G␣ i pathway and a decrease in cAMP, but also G protein-independent, arrestin-mediated cellular responses have been observed (5-7).CRTH2 in particular is expressed on eosinophils, basophils, and T helper type 2 lymphocytes. Activation by PGD 2 or its active metabolites transduces the chemokinetic activity on these immune cells and, by doing so, mediates their recruitment to sites of inflammation (2, 3, 6, 8 -13). In mouse models of allergic asthma or atopic dermatitis, CRTH2 activation promotes eosinophilia and exacerbates pathology (14 -17). In humans, the proinflammatory role of CRTH2 is underscored by the finding that sequence...

In metaphase Xenopus egg extracts, global microtubule growth is mainly promoted by two unrelated microtubule stabilizers, end-binding protein 1 (EB1) and XMAP215. Here, we explore their role and potential redundancy in the regulation of spindle assembly and function. We find that at physiological expression levels, both proteins are required for proper spindle architecture: Spindles assembled in the absence of EB1 or at decreased XMAP215 levels are short and frequently multipolar. Moreover, the reduced density of microtubules at the equator of ⌬EB1 or ⌬XMAP215 spindles leads to faulty kinetochore-microtubule attachments. These spindles also display diminished poleward flux rates and, upon anaphase induction, they neither segregate chromosomes nor reorganize into interphasic microtubule arrays. However, EB1 and XMAP215 nonredundantly regulate spindle assembly because an excess of XMAP215 can compensate for the absence of EB1, whereas the overexpression of EB1 cannot substitute for reduced XMAP215 levels. Our data indicate that EB1 could positively regulate XMAP215 by promoting its binding to the microtubules. Finally, we show that disruption of the mitosis-specific XMAP215-EB1 interaction produces a phenotype similar to that of either EB1 or XMAP215 depletion. Therefore, the XMAP215-EB1 interaction is required for proper spindle organization and chromosome segregation in Xenopus egg extracts. INTRODUCTIONMeiotic and mitotic spindles are microtubule (MT)-based structures that segregate chromosomes during cell division (Karsenti and Vernos, 2001;Scholey et al., 2003;Kwon and Scholey, 2004). The architecture of the spindle is established and maintained by the action of molecular motors and microtubule-associated proteins (MAPs) that organize MTs and regulate their polymerization dynamics (Wittmann et al., 2001;Howard and Hyman, 2007). Interestingly, despite fast turnover of spindle MTs by poleward translocation and dynamic instability of plus ends, the overall length and shape of the spindle are maintained throughout metaphase. Although it is accepted that a balance of MT-stabilizing and -destabilizing activities governs spindle length, it is still not understood how distinct MAPs act collectively to regulate MT dynamic instability (Tournebize et al., 2000;Goshima et al., 2005).XMAP215 is the founding member of the XMAP215/ Dis1/Tog protein family that is conserved from yeast to humans. It increases ϳ7-to 10-fold the MT growth rate in vitro by acting as a processive tubulin polymerase, which while residing on MT ends, supports multiple rounds of addition of individual tubulin dimers (Vasquez et al., 1999;Brouhard et al., 2008). In accordance with the role of XMAP215 as an MT growth promoter, depletion of Dis1/ XMAP215/Tog proteins leads to shorter spindles in Xenopus egg extracts and Drosophila S2 cells as well as to defects in spindle morphology in HeLa cells, Schizosaccharomyces pombe, and Caenorhabditis elegans (Matthews et al., 1998;Tournebize et al., 2000;Garcia et al., 2001;Cassimeris and Morabito, 2004;Goshim...