Regulation of the Dbl proto-oncogene by heat shock cognate protein 70 (Hsc70).
The dbl oncogene product is the defining member of a family of onco-proteins known as Dbl guanine nucleotide exchange factors (GEFs) that facilitate the activation of the small GTP-binding proteins Cdc42, Rac, and Rho. Oncogenic activation of proto-Dbl occurs through loss of the amino-terminal 497 residues, rendering the protein constitutively active. Because both onco- and proto-Dbl contain the structural elements required for GEF activity (i.e. the Dbl homology (DH) and pleckstrin homology (PH) domains), it is thought that the amino terminus of proto-Dbl somehow inhibits the biochemical activity of the protein. To better understand the molecular basis of this regulation, we set forth to identify cellular proteins that preferentially bind the proto-oncogenic form of Dbl. We identified the molecular chaperone heat shock cognate protein (Hsc70) as a binding partner that preferentially interacts with the proto-oncogenic form of Dbl. Dbl is complexed with Hsc70 in transfected cells, as well as in native mouse brain extracts. The interaction between Hsc70 and proto-Dbl is mediated by at least two regions in Dbl, the aminoterminal spectrin homology domain (residues 224-417) and the pleckstrin homology domain (residues 711-808). Overexpression of a dominant negative Hsc70 mutant leads to activation of proto-Dbl GEF activity, indicating that the chaperone negatively regulates proto-Dbl function in vivo. We propose that Hsc70 attenuates Dbl activity by maintaining an inactive conformation in which the amino terminus is "folded over" the catalytic DH-PH domain.
Kauppinen
Krista P
KP
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, USA.
Duan
Faping
F
Wels
Jared I
JI
Manor
Danny
D
D000818:Animals; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D020134:Catalytic Domain; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005799:Genes, Dominant; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006358:Hot Temperature; D047468:Immunoprecipitation; D051379:Mice; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism; D041681:NIH 3T3 Cells; D009705:Nucleosides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015682:Retroviridae Proteins, Oncogenic / Q000096:biosynthesis* / Q000378:metabolism; D013049:Spectrin / Q000737:chemistry; D014162:Transfection
D020662:Guanine Nucleotide Exchange Factors; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494372:Hspa8 protein, mouse; C512843:Mcf2 protein, mouse; D018832:Molecular Chaperones; D009705:Nucleosides; D015682:Retroviridae Proteins, Oncogenic; D013049:Spectrin; D005982:Glutathione Transferase; D020558:GTP Phosphohydrolases