Activation of STAT3 by the Src Family Kinase Hck Requires a Functional SH3 Domain

Steven J Schreiner,Anthony P Schiavone,Thomas E Smithgall

doi:10.1074/jbc.m204255200

Steven J Schreiner, Anthony P Schiavone + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m204255200

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Abstract

STAT3 is a member of a family of transcription factors with Src homology 2 (SH2) domains that are activated by tyrosine phosphorylation in response to a wide variety of cytokines and growth factors. In this study, we investigated the mechanism of STAT3 activation by the Src family of nonreceptor tyrosine kinases, which have been linked to STAT activation in both normal and transformed cell types. Using Sf-9 insect cells, we demonstrate direct STAT3 tyrosine phosphorylation and stimulation of DNA binding activity by five members of the Src kinase family (Src, Hck, Lyn, Fyn, and Fgr). We also observed stable STAT3.Src family kinase complex formation in this system. Recombinant Src family kinase SH3 domains were sufficient for interaction with STAT3, suggesting a mechanistic basis for the Src kinase-STAT3 interaction. To test the contribution of Src family kinase SH3 domains to the recruitment and activation of STAT3 in vivo, we used Rat-2 fibroblasts expressing activated mutants of the myeloid Src family member Hck. Transformation of fibroblasts by an activated Hck mutant lacking the negative regulatory tail tyrosine residue (Hck-YF) induced strong DNA binding activity of endogenous STAT3. Inactivation of Hck SH3 function by Ala replacement of a conserved Trp residue (W93A mutant) completely abolished STAT3 activation by Hck-YF and reduced transforming activity by 50% without affecting Hck kinase activity. Finally, overexpression of STAT3 in Rat-2 cells transiently stimulated Hck and c-Src kinase activity in the absence of extracellular signals, an effect that was dependent upon a putative SH3 binding motif in STAT3. These results support a model in which Src family kinases recruit STAT3 through an SH3-dependent mechanism, resulting in transient kinase activation and STAT3 phosphorylation.

Highlights

) The Src family of protein-tyrosine kinases regulates a diverse array of cellular processes in both normal and transformed cells, including proliferation, survival, differentiation, adhesion and motility [1,2,3,4,5]
To investigate whether Stat3 is a direct substrate for the Src kinase family, c-Src, Hck, Lyn, Fyn and Fgr were co-expressed with Stat3 in Sf-9 insect cells using recombinant baculovirus vectors
The Hck SH3 domain is essential for Stat3 activation in vivo - We investigated the role of Src family kinase SH3 domains in the recruitment and activation of Stat3 in mammalian cells

Summary

EXPERIMENTAL PROCEDURES

Recombinant protein expression in Sf-9 insect cells - Sf-9 cells (InVitrogen) were cultured in Grace’s complete insect cell medium containing 10% fetal bovine serum (FBS) and 50 μg/ml gentamycin. Clarified lysates from Sf-9 cells expressing recombinant Stat were incubated with 10 μg of each GST-SH3 fusion protein or GST alone for 2 h at 4° C. This mutation was combined with Hck-YF by restriction fragment swapping to create Hck-YFW These Hck clones as well as the coding sequences for GFP, Stat, and Stat3-2PA were subcloned into the retroviral expression vector pSRαMSVtkneo [35]. In vitro kinase assay - Rat-2 fibroblasts stably expressing GFP, Stat or Stat3-2PA were infected with c-Src or Hck retroviruses as described above. Kinase activity was assessed 48 h later using the in vitro kinase assay described elsewhere [18,27] This assay employs a 50 kDa GST fusion protein containing residues 331-443 Sam 68 as substrate [38,39] (Santa Cruz Biotechnology) and [(-32P]ATP (New England Nuclear)

RESULTS

DISCUSSION

Cyclin D Hck Actin