A proteomic screen identified stress-induced chaperone proteins as targets of Akt phosphorylation in mesangial cells.
The serine-threonine kinase Akt regulates mesangial cell apoptosis, proliferation, and hypertrophy. To define Akt signaling pathways in mesangial cells, we performed a functional proteomic screen for rat mesangial cell proteins phosphorylated by Akt. A group of chaperone proteins, heat shock protein (Hsp) 70, Hsp90alpha, Hsp90beta, Glucose-regulated protein (Grp) Grp78, Grp94, and protein disulfide isomerase (PDI) were identified as potential Akt substrates by two techniques: (a) in vitro phosphorylation of mesangial cell lysate by recombinant active Akt followed by protein separation by SDS-PAGE or 2-DE and phosphoprotein identification by peptide mass fingerprinting using MALDI-MS, or (b) immunoblot analysis of proteins from PDGF-stimulated mesangial cells using an anti-Akt phospho-motif antibody. In vitro kinase reactions using recombinant proteins confirmed that Akt phosphorylates Hsp70, Hsp90alpha and beta, Grp94, and PDI. Immunoprecipitation of Akt from mesangial cell lysate coprecipitated Grp78 and Hsp70. PDGF stimulation of mesangial cells caused an acidic shift in the isoelectric point of Hsp70, Hsp90, and PDI that was dependent on PI-3K activity for Hsp70 and Hsp90. The data suggest that Akt-mediated phosphorylation of stress-induced chaperones represents a mechanism for regulation of chaperone function during mesangial cell responses to physiologic and pathologic stimuli.
Barati
Michelle T
MT
Department of Medicine, University of Louisville, Louisville, Kentucky 40202, USA.
Rane
Madhavi J
MJ
Klein
Jon B
JB
McLeish
Kenneth R
KR
D000818:Animals; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D003593:Cytoplasm / Q000378:metabolism; D066298:In Vitro Techniques; D050527:Mesangial Cells / Q000201:enzymology / Q000378:metabolism*; D018832:Molecular Chaperones / Q000032:analysis*; D010449:Peptide Mapping; D010766:Phosphorylation; D040901:Proteomics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013312:Stress, Physiological; D013379:Substrate Specificity
D018832:Molecular Chaperones; D011994:Recombinant Proteins; D051057:Proto-Oncogene Proteins c-akt