Paxillin phosphorylation at Ser273 localizes a GIT1–PIX–PAK complex and regulates adhesion and protrusion dynamics

Abstract

Continuous adhesion formation and disassembly (adhesion turnover) in the protrusions of migrating cells is regulated by unclear mechanisms. We show that p21-activated kinase (PAK)–induced phosphorylation of serine 273 in paxillin is a critical regulator of this turnover. Paxillin-S273 phosphorylation dramatically increases migration, protrusion, and adhesion turnover by increasing paxillin–GIT1 binding and promoting the localization of a GIT1–PIX–PAK signaling module near the leading edge. Mutants that interfere with the formation of this ternary module abrogate the effects of paxillin-S273 phosphorylation. PAK-dependent paxillin-S273 phosphorylation functions in a positive-feedback loop, as active PAK, active Rac, and myosin II activity are all downstream effectors of this turnover pathway. Finally, our studies led us to identify in highly motile cells a class of small adhesions that reside near the leading edge, turnover in 20–30 s, and resemble those seen with paxillin-S273 phosphorylation. These adhesions appear to be regulated by the GIT1–PIX–PAK module near the leading edge.

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

“…We have previously shown that SLK is required for efficient focal adhesion turnover [78]. Furthermore, we have determined that SLK is capable of localizing with paxillin (Figure 3.1), a focal adhesion protein that has also been implicated in the regulation of adhesion turnover, to the leading edge and membrane ruffle of migrating cells [78,99,107,120]. Since SLK is required for fusion in C2C12, a process whereby coordinated migration signaling is required for the alignment and formation of myoblasts into myotubes, we tested for, and observed that SLK is capable of co-localizing with paxillin in differentiating myoblasts (Figure 3.1) [129,130].…”

“…For example, the combination of adhesion and angiotensin II stimulation has been shown to result in the phosphorylation of paxillin LIM2 T396/401 and LIM3 S455/479, which is believed to target paxillin to sites of focal adhesions [99]. Although LIM2 has been shown to associate and co-immunoprecipitate with a kinase that can phosphorylate threonines 396/401 in human cells and LIM3 to bind a detergent-insoluble kinase that can phosphorylate serines 455/479, when tested, these kinases were immune to inhibition by a variety of broad-spectrum serine/threonine kinase inhibitors, suggesting that the identity of these kinases are novel in nature [66,86,120]. Despite this however, several candidate serine/threonine kinases have been identified for paxillin including: protein kinase C, MAPK, JNK, ERK1/2, the p21-activated protein kinase PAK3 and the integrin-linked kinase, ILK [121,122].…”

Dynamic Phosphorylation of Tyrosine 665 in Pseudopodium-enriched Atypical Kinase 1 (PEAK1) Is Essential for the Regulation of Cell Migration and Focal Adhesion Turnover

“…Currently, ϳ180 identified proteins comprise the adhesome including paxillin, Src, FAK, and ␣-actinin (30,31). Paxillin enters FAs early in assembly and is well established as an FA marker (11,20,32,33). As assembly progresses, scaffolds such as ␣-actinin incorporate, marking FA maturation (11).…”

Regulation of actin cytoskeleton dynamics by Arf-family GTPases

“…Localization to focal adhesions is largely mediated by interaction of the C-terminal domain of GIT with paxillin [63][64][65]. Both GIT1 and GIT2 also bind constitutively to the Rac and Cdc42 GEF PIX (Pak-interacting exchange factor), and PIX in turn binds the serine-threonine kinase PAK (p21-activated kinase).…”

“…In Rat2 fibroblasts, knockdown of GIT1 reduced protrusiveness and decreased the rate of focal adhesion turnover. Moreover, reconstitution experiments indicated that the catalytic activities of both β-PIX and PAK were necessary to restore normal turnover kinetics [65]. However, in HeLa cells, knockdown of GIT1 was found to have no observable effect, whereas knockdown of GIT2 actually enhanced focal adhesion turnover and lamellipodial extension [71].…”