p50(cdc37) acting in concert with Hsp90 is required for Raf-1 function

Area of Science:

• Molecular Biology
• Cell Signaling
• Biochemistry

Background:

• p50(cdc37) is a known component of the sevenless receptor/mitogen-activated protein kinase (MAPK) pathway, but its specific function and targets remain undefined.
• The role of chaperone proteins, like Heat Shock Protein 90 (Hsp90), in regulating signaling pathways is increasingly recognized.

Purpose of the Study:

• To biochemically investigate the function of p50(cdc37) and its partner Hsp90 in the Raf/Mek/MAPK signaling pathway.
• To elucidate the mechanism by which p50(cdc37) influences Raf-1 activation and downstream signaling.

Main Methods:

• Coexpression of wild-type and mutant p50(cdc37) with Raf-1 in Sf9 cells.
• Biochemical assays to assess Raf-1 activation and Hsp90 recruitment.
• Inhibition studies using geldanamycin (GA), an Hsp90-specific inhibitor.

Main Results:

• Coexpression of p50(cdc37) with Raf-1 robustly activated Raf-1 kinase.
• p50(cdc37) is the primary determinant for Hsp90 recruitment to Raf-1.
• Inhibition of the p50(cdc37)-Hsp90 complex formation by a p50(cdc37) mutant or GA blocked Raf-1 and MAPK activation.

Conclusions:

• Formation of a ternary complex involving Raf-1, p50(cdc37), and Hsp90 is critical for Raf-1 activity.
• The p50(cdc37)-Hsp90 complex is essential for regulating Raf-1 kinase function and MAPK pathway signaling.
• This study provides the first biochemical evidence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation.