A Proteomic Screen Identified Stress-Induced Chaperone Proteins as Targets of Akt Phosphorylation in Mesangial Cells

Abstract

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, Hsp90α, Hsp90β, 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, Hsp90α and β, Grp94, and PDI. Immunoprecipitation of Akt from mesangial cell lysate co-precipitated 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 stressinduced chaperones represents a mechanism for regulation of chaperone function during mesangial cell responses to physiologic and pathologic stimuli.

Hsp90 phosphorylation, Wee1 and the cell cycle

“…27,35 Hsp90 is also a substrate for DNA-dependent protein kinase, Akt, B-Raf and casein kinase II (CKII). 36,37,40,41 Further, PKA phosphorylation of Thr90 induced by 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitors has been reported to increase association of human Hsp90α with the client protein eNOS. 42 Lastly, a study reported that protein phosphatase 5 (Pp5/Ppt1) can dephosphorylate Hsp90 in vitro.…”

Epigallocatechin-3-gallate (EGCG) attenuates inflammation in MRL/lpr mouse mesangial cells

“…The effect of EGCG on Akt is important in the context of SLE as Akt activation has been shown to enhance mesangial cell proliferation, hypertrophy, migration, and extracellular matrix protein production which can all contribute to glomerular injury. 51,52 Akt is a serine/threonine kinase (also known as protein kinase B) that plays a key role in cellular metabolism, growth and survival. 53 Although Akt is activated by phosphorylation of both Thr308 and Ser473 residues, the Ser473 phosphorylation site is considered key as it stabilizes the active conformational state of Akt.…”

Hamartin-Hsp70 Interaction Is Necessary for Akt-Dependent Tuberin Phosphorylation during Heat Shock

“…22) Furthermore, Akt and tuberin phosphorylation was also inhibited by KNK437, an Hsp70 inhibitor. Considering these data and the report that Hsp70 binds Akt, 23) we conclude that Akt activity is regulated by the binding of Hsp70, and that this interaction further influences The cells were then exposed to HS at 42 C for 30 min before harvest. Cell extracts were subjected to immunoblot with the indicated antibodies.…”