The Tyrosine Kinase Pyk2 Regulates Arf1 Activity by Phosphorylation and Inhibition of the Arf-GTPase-activating Protein ASAP1

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

Src-family tyrosine kinase fyn phosphorylates phosphatidylinositol 3-kinase enhancer-activating Akt, preventing its apoptotic cleavage and promoting cell survival

“…PIKE-A possesses similar structural domains as ASAP1 (ArfGAP containing SH3, ankyrin repeats and PH domain), which prominently binds to both FAK and pyk2 (proline-rich tyrosine (y) kinase 2), a nonreceptor tyrosine kinase. 21,22 Unfortunately, PIKE-A does not bind to either tyrosine kinase (data not shown). ASAP1 interacts with FAK or pyk2 through its SH3 domain, however, PIKE-A does not contain any SH3 domain.…”

“…Pyk2-phosphorylating ASAP1 affects its phosphoinositides binding profiles and reduces its ArfGAP activity. 21 Our previous study demonstrates that PIKE-A binds a variety of phosphatidylinositol lipids and possesses robust GTPase activity. 23 It remains unknown whether fyn-mediated tyrosine phosphorylation on PIKE-A also influences the above biochemical effects.…”

Regulation of actin cytoskeleton dynamics by Arf-family GTPases

“…The ASAPs interact with several focal adhesion components, including Src, FAK (focal adhesion kinase), Pyk2 (proline-rich tyrosine kinase 2) and paxillin, and localize to focal adhesions or focal complexes in many cell types (Figure 4). ASAPs are phosphorylated by Src, FAK and Pyk2, and phosphorylation by Pyk2 has been shown to inhibit Arf GAP activity [58]. ASAP1 function appears to be important for focal adhesion dynamics, because both overexpression and knockdown have been shown to affect FA assembly [59,60].…”

Calcium-dependent growth regulation of small cell lung cancer cells by neuropeptides

“…It was suggested that PSGAP may be an essential protein for regulation of cytoskeletal organization by Pyk2 via RhoGTPases (Ren et al 2001). Furthermore, Pyk2 binds to an ArfGTPase-activating protein ASAP1, thereby regulating Arf1 activity by phosphorylation building another bridge to reorganization of the actin cytoskeleton (Kruljac-Letunic et al 2003). Pyk2 contains a consensus paxillin binding sequence within its C-terminus and it has been shown that Pyk2 can associate with paxillin or paxillin-related proteins Hic-5 or leupaxin (Schlaepfer et al 1999).…”