Biochemical properties of the Cdc42-associated tyrosine kinase ACK1. Substrate specificity, authphosphorylation, and interaction with Hck.
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 Tyr284 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.
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001549:Benzamides; D015153:Blotting, Western; D019556:COS Cells; D020134:Catalytic Domain; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D000068877:Imatinib Mesylate; D007313:Insecta; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry; D008957:Models, Genetic; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016315:Proto-Oncogene Proteins c-abl / Q000737:chemistry; D051572:Proto-Oncogene Proteins c-hck; D011743:Pyrimidines / Q000494:pharmacology; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry*; D018909:src Homology Domains
D001549:Benzamides; D004791:Enzyme Inhibitors; D008024:Ligands; D010455:Peptides; D010879:Piperazines; D011518:Proto-Oncogene Proteins; D011743:Pyrimidines; C011083:polyproline; D014443:Tyrosine; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D016315:Proto-Oncogene Proteins c-abl; D051572:Proto-Oncogene Proteins c-hck; D020764:cdc42 GTP-Binding Protein