Methods Enzymol. 2006;406:250-60.

Purification and enzyme activity of ACK1.

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Department of Physiology and Biophysics, SUNY at Stony Brook, NY, USA.

Abstract

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.

PMID:: 16472662; [PubMed - indexed for MEDLINE]

Publication Types, MeSH Terms, Substances, Grant Support

Publication Types

Research Support, N.I.H., Extramural

MeSH Terms

Substances

Grant Support

CA 28146/CA/NCI NIH HHS/United States

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