Novel families of oncogenic tyrosine kinases
Our laboratory is investigating other families of nonreceptor tyrosine kinases in which the mechanisms for regulation and substrate recognition are not as well understood. Our starting point for these projects is to express the kinases using the baculovirus/Sf9 system, purify them, and study their enzymatic properties. We are particularly interested in understanding how the noncatalytic regions of the enzymes participate in kinase function.
Brk: Brk (breast tumor kinase) was identified by Mark Crompton's group in a study of kinase expression in human metastatic breast tumors. Brk expression was low or undetectable in normal mammary tissues or in benign lesions. However, approximately two-thirds of the breast tumors that were examined overexpressed Brk. Brk is a member of the Frk family of nonreceptor tyrosine kinases. Brk possesses SH3, SH2, and catalytic domains in a similar arrangement to that of Src family kinases. Using purified Brk, we showed that the enzyme is regulated by autophosphorylation at Tyr342 and by intramolecular interactions involving the SH3 and SH2 domains. We used a proteomic strategy to identify Brk-interacting partners.
For more details, see:
H. Qiu & W.T. Miller (2002). Regulation of the nonreceptor tyrosine kinase Brk by autophosphorylation and by autoinhibition. J. Biol. Chem. 277, 34634-34641.
H. Qiu & W.T. Miller (2004). Role of the Brk SH3 domain in substrate recognition. Oncogene 23, 2216-2223.
H. Qiu, F. Zappaosta, W. Su, R.S. Annan, and W.T. Miller (2005). Interaction between Brk kinase and insulin receptor substrate-4. Oncogene 24, 5656-5664.
ACK1: ACK1 is a nonreceptor tyrosine kinase that is a downstream target of Cdc42. We purified the enzyme using the Sf9/baculovirus system & studied its enzymatic properties. Ack autophosphorylates at Tyr284. It binds to Src-kinases and is phosphorylated by them in COS cells. We recently found that ACK1 has the capacity to phosphorylate serine as well as tyrosine residues in substrates; this dual specificity appears to be unique among nonreceptor tyrosine kinases.
For more details, see:
N. Yokoyama & W.T. Miller (2003). Biochemical properties of the Cdc42-associated tyrosine kinase ACK1: substrate specificity, autophosphorylation, and interaction with Hck. J. Biol. Chem. 278, 47713-47723.
N. Yokoyama, J. Lougheed, and W.T. Miller (2005). Phosphorylation of WASP by the Cdc42-associated kinase ACK1: Dual hydroxyamino acid specificity in a tyrosine kinase. J. Biol. Chem 280, 42219-42226.