Phosphorylation of WASP by the Cdc42-associated Kinase ACK1

Abstract

ACK1 is a nonreceptor tyrosine kinase that associates specifically with Cdc42. Relatively few ACK1 substrates and interacting proteins have been identified. In this study, we demonstrated that ACK1 phosphorylates the Wiskott-Aldrich syndrome protein (WASP), a Cdc42 effector that plays an important role in the formation of new actin filaments. ACK1 and WASP interact in intact cells, and overexpression of ACK1 promotes WASP phosphorylation. Phosphorylation of WASP in vitro was enhanced by the addition of Cdc42 or phosphatidylinositol 4,5-biphosphate, presumably due to release of the autoinhibitory interactions in WASP. Surprisingly, when we mapped the sites of WASP phosphorylation, we found that ACK1 possesses significant serine kinase activity toward WASP (directed at Ser-242), as well as tyrosine kinase activity directed at Tyr-256. A serine peptide derived from the Ser-242 WASP phosphorylation site is also a substrate for ACK1. ACK1 expressed in bacteria retained its serine kinase activity, eliminating the possibility of contamination with a copurifying kinase. Serine phosphorylation of WASP enhanced the ability of WASP to stimulate actin polymerization in mammalian cell lysates. Thus, the tyrosine kinase ACK1 acts as a dual specificity kinase toward this substrate. In contrast to other dual specificity kinases that more closely resemble Ser/Thr kinases, ACK1 is a tyrosine kinase with an active site that can accommodate both types of hydroxyamino acids in substrates.The nonreceptor tyrosine kinase ACK1 2 (activated Cdc42-associated kinase-1) is a specific effector kinase for the GTP-bound form of Cdc42. ACK1 is a member of a distinct family of nonreceptor tyrosine kinases that includes ACK1, ACK2, and TNK in mammals and homologs in Drosophila and Caenorhabditis elegans (1, 2). The catalytic domain sequences of ACK family kinases are characteristic of tyrosine kinases, yet they are divergent from most other families of nonreceptor tyrosine kinases; they are most closely related to the epidermal growth factor receptor family kinases (3, 4). ACK kinases also have a unique structural organization; C-terminal to the catalytic domain, they possess SH3 domains, Cdc42-binding CRIB domains, and polyproline regions. The placement of the SH3 domain C-terminal to the catalytic domain is different from the other families of nonreceptor tyrosine kinases. ACK kinases are activated by cell adhesion, and they play important roles in cell spreading and motility (5-7). Recent work has also focused on the involvement of ACK kinases in signaling pathways leading to clathrin-mediated receptor endocytosis (8, 9).Relatively little is known about the physiological substrates of ACK kinases. Upon epidermal growth factor stimulation, ACK2 binds and phosphorylates sorting nexin 9 (SH3PX1), which promotes epidermal growth factor receptor degradation (9). In hematopoietic cells, ACK1 binds and phosphorylates the adaptor protein HSH2 (10). For both HSH2 and SH3PX1, ACK substrate recognition is governed by interactions with the noncatalytic r...

c-Abl-mediated Phosphorylation of WAVE3 Is Required for Lamellipodia Formation and Cell Migration

“…Phosphorylation of N-WASP/WASP proteins by non-receptor tyrosine kinases, such as ACK1 enhances the ability of WASP to stimulate actin polymerization (22), whereas growth factor stimulation of cultured cells results in hyper-phosphorylation of the WAVE proteins as well as a delay in their mobility in SDS-PAGE (23). WAVE1 was shown to be phosphorylated by the cyclindependent kinase 5 (24), whereas phosphorylation of WAVE2 has recently been shown to involve the non-receptor tyrosine kinase c-Abl (25), resulting in both cases in the stimulation of membrane ruffling and cell spreading.…”

The actin cytoskeleton coordinates the signal transduction and antigen processing functions of the B cell antigen receptor

“…The actin nucleation promoting activity of WASP is increased by phosphorylation at Y256, Y291, S242, S483 and S484 sites of WASP (Cory et al, 2002, 2003; Yokoyama et al, 2005), which stabilizes its open active conformation. We have shown that BCR activation increases the levels of phosphorylated WASP and recruits phosphorylated WASP to the membrane of B cells at the contact zone in a Btk-dependent manner (Sharma et al, 2009; Liu et al, 2011).…”

“…However, whether Btk can directly phosphorylate WASP is not known. It has been suggested that WASP is a substrate of the Src family kinase Hck (Cory et al, 2002) and activated Cdc42-associated kinase 1 (ACK1) (Yokoyama et al, 2005), but whether these kinases are responsible for WASP phosphorylation in B cells and their relationship with Btk have not yet been determined. The actin binding ability of ezrin is controlled by phosphorylation at T567 (Table 1).…”

Surfing the big WAVE: Insights into the role of WAVE3 as a driving force in cancer progression and metastasis

“…The non-receptor tyrosine kinase ACK1 phosphorylates and enhances the WASP-mediated actin polymerization [70], while stimulation of cultured cells with growth factors increases the phosphorylation levels of the WAVE proteins [71]. Phosphorylation of WAVE1 by CDK5 [72], or of WAVE2 by the non-receptor tyrosine kinase c-Abl [73], leads to the stimulation of membrane ruffling and cell spreading [74].…”