PIAS1-mediated Sumoylation of Focal Adhesion Kinase Activates Its Autophosphorylationn

Abstract

Focal adhesion kinase (FAK) is a protein tyrosine kinase enriched in focal adhesions, which plays a critical role in integrin-dependent cell motility and survival. The crucial step in its activation is autophosphorylation on Tyr-397, which promotes the recruitment of several enzymes including Src family kinases and the activation of multiple signaling pathways. We found in a yeast two-hybrid screen that the N-terminal domain of FAK interacted with protein inhibitor of activated STAT1 (PIAS1). This interaction was confirmed and shown to be direct using in vitro assays. PIAS1 was co-immunoprecipitated with FAK from transfected cells and brain extracts. PIAS1 has recently been recognized as a small ubiquitin-like modifier (SUMO) ligase. In the presence of PIAS1 and SUMO-1, FAK was sumoylated in intact cells, whereas PYK2, a closely related enzyme, was not. Sumoylation occurred on Lys-152, a residue conserved in FAK during evolution. Sumoylated FAK, like PIAS1, was recovered predominantly from the nuclear fraction. Sumoylation did not require the catalytic activity or autophosphorylation of FAK. In contrast, sumoylation increased dramatically the ability of FAK to autophosphorylate in intact cells and in immune precipitate kinase assays. Endogenous FAK was sumoylated in the presence of PIAS1 and SUMO-1 independently of cell adhesion, and autophosphorylation of sumoylated FAK was persistently increased in suspended cells. These observations show that sumoylation controls the activity of a protein kinase and suggest that FAK may play a novel role in signaling between the plasma membrane and the nucleus.Focal adhesion kinase (FAK) 1 is a non-receptor cytoplasmic tyrosine kinase of 125 kDa (1, 2) implicated in integrin-mediated signal transduction (reviewed in Refs. 3-5). It is also activated by a variety of extracellular stimuli including G protein-coupled receptors and growth factor receptors (see Refs. 5 and 6). In adherent mammalian cells in culture FAK is located in focal adhesions (1, 2) and appears important for the regulation of their turnover (7). FAK is critical for adhesion-dependent cell survival (8) and integrin-mediated motility (9). Its physiological importance is demonstrated by the embryonic mortality of FAK knockout mice (7), whereas recent work underlines its role in tumor invasiveness (10). Autophosphorylation at Tyr-397 is a crucial event for FAK biological function, because it creates a high affinity binding site for proteins harboring Src homology 2 (SH2) domains, such as Src and Fyn (11), Grb7 (12), and phosphatidylinositide 3Ј-OH-kinase, which activates the anti-apoptotic Akt pathway (13,14). Following their binding to phospho-Tyr-397, Src or Fyn phosphorylate FAK at other tyrosine residues (15, 16) as well as associated proteins, thereby leading to the activation of several signaling pathways (see 5).In contrast to the recent progress in elucidating the signaling pathways downstream from FAK, relatively little is known about the molecular mechanisms regulating FAK activity. The tyrosine kinase ...

Sumoylated RGS-Rz Proteins Act as Scaffolds for Mu-Opioid Receptors and G-Protein Complexes in Mouse Brain

“…The machinery necessary for protein sumoylation is mostly nuclear (Melchior et al, 2003), although an increasing number of sumoylated proteins act outside the nucleus. These include cytosolic proteins such as a-catenin, SCG10, phosducin (Gocke et al, 2005;Klenk et al, 2006), and the membrane spanning or attached proteins GLUT1 and GLUT4 transporters (Giorgino et al, 2000), or the focal adhesion kinase (Kadaré et al, 2003). It is therefore possible that these proteins are subject to nucleocytoplasmic cycling that facilitates their sumoylation in the nucleus and the subsequent redistribution to the cytosol and/or cell membrane.…”

A single structurally conserved SUMOylation site in CRMP2 controls NaV1.7 function

“…26 Previous work has indicated that phosphorylation and SUMOylation can interact within the same protein to alter prevalence of the other modification. Examples of this include focal adhesion kinase where SUMOylation promotes autophosphorylation, 40 heat-shock factors 1 and 4b, myocyte enhancer factor 2, GluK2 kainate receptor, or estrogen receptor b that require phosphorylation for secondary SUMOylation, 41-45 DNA binding protein SATB1 where phosphorylation prevents SUMOylation, 46 and microtubule stabilizing Tau protein where either phosphorylation or SUMOylation promotes the other modification. 47 In the case of CRMP2, Fyn phosphorylation reduces SUMOylation, consequently reducing CRMP2s interaction with NaV1.7.…”

SUMOylation-regulated Protein Phosphorylation, Evidence from Quantitative Phosphoproteomics Analyses

“…Using FAK, a non-receptor tyrosine kinase, as a specific example of tyrosine kinases, we demonstrated that SUMOylation reduction decreased the activity of FAK. The kinase activity of FAK is dependent on its autophosphorylation on Tyr-397, which, in turn, is positively modulated by SUMOylation (13). We examined whether GA treatment could alter FAK autophosphorylation at Tyr-397 (as a readout of the activation of FAK).…”