Dual roles of the SH3 and SH2 domains of Src family kinases

For a general review of our work on Src kinases, see Accounts of Chemical Research (2003) 36, 393-400.

In addition to their tyrosine kinase catalytic domains (blue), Src family kinases possess amino-terminal regulatory regions termed SH3 domains (yellow) and SH2 domains (green). These modular domains mediate intramolecular and intermolecular interactions that are important in signal transduction. For Src-family tyrosine kinases, the SH3 and SH2 domains are important in maintaining an inactive conformation. The three-dimensional structure of the Src-kinase Hck is shown below. The structure was determined by Dr. John Kuriyan and his colleagues (Mol. Cell 3: 639-648).


SH2 and SH3 domains play two important roles in substrate recognition by nonreceptor tyrosine kinases:

1. Activation of Src-family tyrosine kinases. We showed that the SH3 domains of Src family kinases have a major regulatory role in controlling tyrosine kinase activity. For example, binding of the HIV-1 protein Nef to the SH3 domain of the Src-family kinase Hck activates the enzyme:

For more details, see:

I. Moarefi, M. LaFevre-Bernt, F. Sicheri, M. Huse, C.-H. Lee, J. Kuriyan, and W.T. Miller (1997). Activation of the Src-Family Tyrosine Kinase Hck by SH3 Domain Displacement. Nature 385, 650-653.

M. LaFevre-Bernt, F. Sicheri, A. Pico, M. Porter, J. Kuriyan, & W.T. Miller (1998). Intramolecular regulatory interactions in the Src family kinase Hck probed by mutagenesis of a conserved tryptophan residue. J. Biol. Chem. 273, 32129-32134.

M. Porter, T. Schindler, J. Kuriyan, and W.T. Miller (2000). Reciprocal regulation of Hck activity by phosphorylation of Tyr527 and Tyr416. J. Biol. Chem. 275, 2721-2726.

2. SH2 and SH3 domains assist tyrosine kinases in recognizing cellular substrates. Many of the best substrates for Src kinases contain ligands for the SH3 and/or SH2 domains. Binding promotes phosphorylation by the catalytic domain; in this way, kinase activation is coupled to substrate recognition.

For details on this work, please see:

P. Pellicena, K.R. Stowell, and W.T. Miller (1998). Enhanced phosphorylation of Src-family kinase substrates containing SH2 domain binding sites. J. Biol. Chem. 273, 15325-15328.

M.P. Scott, F. Zappacosta, E.Y. Kim, R.S. Annan, & W.T. Miller (2002). Identification of novel SH3 domain ligands for the Src family kinase Hck: WASP, WIP, and ELMO1. J. Biol. Chem. 277, 28238-28346.\

S.S. Yadav and W.T. Miller (2008). The evolutionarily conserved arrangement of domains in Src family kinases is important for substrate recognition. Biochemistry 47, 10871-10880.

A related area of interest is processive phosphorylation. Many Src substrates contain multiple sites for phosphorylation. We showed that Src can phosphorylate these substrates by a processive mechanism, in which the kinase remains bound to the substrate while catalyzing multiple rounds of phosphorylation. We are focusing on processive phosphorylation of p130Cas, a Src substrate that resides in focal adhesions. For more details on this work, see:

M.P. Scott and W.T. Miller (2000). A peptide model system for processive phosphorylation by Src-family kinases. Biochemistry 39, 14531-14537.

P. Pellicena and W.T. Miller (2001). Processive phosphorylation of p130Cas by Src is dependent on SH3-polyproline interactions. J. Biol. Chem 276, 28190-28196.

G.S. Goldberg, D.B. Alexander, P. Pellicena, Z.-Y. Zhang, H. Tsuda, & W.T. Miller (2003). Src phosphorylates Cas on tyrosine 253 to promote migration of transformed cells. J. Biol. Chem 278, 46533-46540.

P. Patwardhan, Y. Shen, G. Goldberg, and W.T. Miller (2006). Individual Cas phosphorylation sites are dispensable for processive phosphorylation by Src and anchorage-independent cell growth. J. Biol. Chem 281, 20689-20697.

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.


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.

V. Prieto-Echagüe, A. Gucwa, B.P. Craddock, D.A. Brown, and W.T. Miller (2010). Cancer-associated mutations activate the nonreceptor tyrosine kinase Ack1. J. Biol. Chem. 285, 10605-10615.

V. Prieto-Echagüe, A. Gucwa, D.A. Brown, and W.T. Miller (2010). Regulation of Ack1 localization and activity by the amino-terminal SAM domain. BMC Biochemistry 11, 42.