Biochemical Properties of the Cdc42-associated Tyrosine Kinase ACK1

Abstract

ACK1 (activated Cdc42-associated kinase 1) is a nonreceptor tyrosine kinase and the only tyrosine kinase known to interact with Cdc42. To characterize the enzymatic properties of ACK, we have expressed and purified active ACK using the baculovirus/Sf9 cell system. This ACK1 construct contains (from N to C terminus) the kinase catalytic domain, SH3 domain, and Cdc42-binding Cdc42/Rac interactive binding (CRIB) domain. We characterized the substrate specificity of ACK1 using synthetic peptides, and we show that the specificity of the ACK1 catalytic domain most closely resembles that of Abl. Purified ACK1 undergoes autophosphorylation, and autophosphorylation enhances kinase activity. We identified Tyr 284 in the activation loop of ACK1 as the primary autophosphorylation site using mass spectrometry. When expressed in COS-7 cells, the Y284F mutant ACK1 showed dramatically reduced levels of tyrosine phosphorylation. Although the SH3 and CRIB domains of purified ACK1 are able to bind ligands (a polyproline peptide and Cdc42, respectively), the addition of ligands did not stimulate tyrosine kinase activity. To characterize potential interacting partners for ACK1, we screened several SH2 and SH3 domains for their ability to bind to full-length ACK1 or to the catalytic-SH3-CRIB construct. ACK1 interacts most strongly with the SH3 domains of Src family kinases (Src or Hck) via its C-terminal proline-rich domain. Co-expression of Hck with kinase-inactive ACK1(K158R) in mammalian cells resulted in tyrosine phosphorylation of ACK1, suggesting that ACK1 is a substrate for Hck. Our data suggest that Hck is a novel binding partner for ACK1 that can regulate ACK1 activity by phosphorylation.Members of the Rho family of small GTP-binding proteins couple extracellular signals to the regulation of cell morphology, adhesion, differentiation, and proliferation (1-5). A number of proteins have been identified as targets for the Rho family proteins Rac and/or Cdc42, including p21-activated kinases (6 -8), Wiscott-Aldrich syndrome proteins (9 -11), mixed lineage kinases (12), and IQGAP (13-16). The ACK family nonreceptor tyrosine kinases (ACK1 and ACK2) associate specifically with Cdc42 and act as Cdc42 effectors in several signaling pathways (6,17,18). ACK1 has been reported to phosphorylate Dbl, a guanine nucleotide exchange factor toward Rho family proteins, thereby promoting Dbl activity. In this way, ACK1 is thought to act as a mediator of EGF 1 signals to Rho family GTP-binding proteins (19). ACK2 mediates cell adhesion signals initiated by integrin ␤ 1 in a Cdc42-dependent manner (20). ACKs interact directly with the clathrin heavy chain and participate in the regulation of receptor-mediated endocytosis (21, 22). However, the physiological functions as well as specific target molecules of ACKs are still incompletely understood.The domain structure of ACK kinases consists of an Nterminal tyrosine kinase catalytic domain followed by an SH3 domain, a Cdc42/Rac interactive binding (CRIB) domain, and a proline-rich region (see Fig. 1...

Crystal Structures of the Phosphorylated and Unphosphorylated Kinase Domains of the Cdc42-associated Tyrosine Kinase ACK1

“…In molecule A, clear electron density was observed for a phosphorylated tyrosine in the activation loop (Tyr 284 ), whereas no evidence of phosphorylation was observed in molecule B. Importantly, Tyr 284 is the only tyrosine in the activation loop and is the primary site of autophosphorylation in ACK1K (19). Examination of crystal contacts near the activation loop of molecule B suggests that the phosphorylated activation loop is prohibited from incorporating into the crystal lattice at this position because of small changes in amino acid conformations in the loop.…”

“…Structural studies have revealed that the molecular basis for this activation in PAK1 involves release of an intramolecular autoinhibitory module upon Cdc42 binding (17). ACK kinases appear functionally similar to PAK kinases in cell-based experiments, in which Cdc42 binding promotes ACK1 activation (8,18,19). However, as the sequence characteristics and domain organization around the CRIB motif are dissimilar in ACK and PAK1, the molecular details of ACK activation by Cdc42 are likely to be different.…”

“…However, as the sequence characteristics and domain organization around the CRIB motif are dissimilar in ACK and PAK1, the molecular details of ACK activation by Cdc42 are likely to be different. Additionally, unlike members of the PAK family, Cdc42 is unable to promote the activation of ACKs in vitro (2,19), suggesting that other cellular components may be involved. The ACKs are also the only tyrosine kinases with an SH3 domain C-terminal to the kinase domain.…”

“…The Src family tyrosine kinases, including Src and Hck, and the Abl tyrosine kinase have SH3 domains (located N-terminal to the kinase domain) that serve as autoinhibitory modules of the unphosphorylated kinase. These kinases can be activated by binding of a protein or peptide to the SH3 domain (20 -23); however, an SH3 ligand does not increase ACK1 catalytic activity in vitro (19), suggesting that the SH3 domain does not directly regulate the enzymatic activity of ACK1. The SH3 domain might alternatively play a role in protein localization or in substrate re- The atomic coordinates and structure factors (code 1U46, 1U54, and 1U4D) 1 The abbreviations used are: SH3, Src homology 3; EGFR, epidermal growth factor receptor; ACK1K, ACK1 kinase domain; AMP-PCP, adenosine 5Ј-(␤,␥-methylenetriphosphate); r.m.s.d., root mean square deviation; EGFRK, epidermal growth factor receptor kinase domain; CRIB, cdc42/Rac interactive binding domain.…”

“…The Src family tyrosine kinases, including Src and Hck, and the Abl tyrosine kinase have SH3 domains (located N-terminal to the kinase domain) that serve as autoinhibitory modules of the unphosphorylated kinase. These kinases can be activated by binding of a protein or peptide to the SH3 domain (20 -23); however, an SH3 ligand does not increase ACK1 catalytic activity in vitro (19), suggesting that the SH3 domain does not directly regulate the enzymatic activity of ACK1. The SH3 domain might alternatively play a role in protein localization or in substrate re-cruitment as in other proteins (24,25).…”