Purification and enzyme activity of ACK1.
The activated Cdc42 associated kinases (ACKs) are nonreceptor tyrosine kinases that are specific targets of Cdc42. To study the biochemical properties of ACK1, we expressed and purified the enzyme using the baculovirus/Sf9 cell system. This ACK1 construct contains (from N- to C-terminus) the kinase catalytic domain, SH3 domain, and Cdc42-binding CRIB domain. We describe enzyme activity assays based on synthetic peptide substrates. The best such substrate is a peptide derived from the site of ACK1-catalyzed phosphorylation of the Wiskott-Aldrich syndrome protein (WASP). Although the SH3 and CRIB domains of purified ACK1 are able to bind ligands (a polyproline peptide and Cdc42, respectively), the ligands did not stimulate in vitro tyrosine kinase activity. Purified ACK1 undergoes autophosphorylation at Tyr284, and autophosphorylation increases kinase activity.
Department of Physiology and Biophysics, SUNY at Stony Brook, NY, USA.
Noriko
N
Yokoyama
W Todd
WT
Miller
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D018411:Spodoptera; D013379:Substrate Specificity
D009842:Oligopeptides; C498594:Tnk2 protein, mouse; D011505:Protein-Tyrosine Kinases; C403262:TNK2 protein, human