title: Pnck induces ligand-independent EGFR degradation by probable perturbation of the Hsp90 chaperone complex.
abstract: We have recently described a novel role for pregnancy-upregulated non-ubiquitous calmodulin kinase (Pnck) in the induction of ligand-independent epidermal growth factor receptor (EGFR) degradation (Deb TB, Coticchia CM, Barndt R, Zuo H, Dickson RB, and Johnson MD. Am J Physiol Cell Physiol 295: C365-C377, 2008). In the current communication, we explore the probable mechanism by which Pnck induces ligand-independent EGFR degradation. Pnck-induced EGFR degradation is calcium/calmodulin independent and is regulated by cell density, with the highest EGFR degradation observed at low cell density. Pnck is a novel heat shock protein 90 (Hsp90) client protein that can be co-immunoprecipitated with Hsp90. Treatment of Pnck-overexpressing cells with the pharmacologic Hsp90 inhibitor geldanamycin results in enhanced EGFR degradation, and destruction of Pnck. In cells in which Pnck is inducing EGFR degradation, we observed that Hsp90 exhibits reduced electrophoretic mobility, and through mass spectrometric analysis of immunopurified Hsp90 protein we demonstrated enhanced phosphorylation at threonine 89 and 616 (in both Hsp90-α and -β) and serine 391 (in Hsp90-α). Kinase-active Pnck protein is degraded by the proteasome, concurrent with EGFR degradation. A Pnck mutant (T171A) protein with suppressed kinase activity induced EGFR degradation to essentially the same level as wild-type (WT) Pnck, suggesting that Pnck kinase activity is not required for the induction of EGFR degradation. Although EGFR is degraded, overexpression of WT Pnck paradoxically promoted cellular proliferation, whereas cells expressing mutant Pnck (T171A) were growth inhibited. WT Pnck promoted S to G(2) transition, but cells expressing the mutant exhibited higher residency time in S phase. Basal MAP kinase activity was inhibited by WT Pnck but not by mutant T171A Pnck protein. Cyclin-dependent kinase (Cdk) inhibitor p21/Cip-1/Waf-1 was transcriptionally suppressed downstream to MAP kinase inhibition by WT Pnck, but not the mutant protein. Collectively, these data suggest that 1) Pnck induces ligand-independent EGFR degradation most likely through perturbation of Hsp90 chaperone activity due to Hsp90 phosphorylation, 2) EGFR degradation is coupled to proteasomal degradation of Pnck, and 3) modulation of basal MAP kinase activity, p21/Cip-1/Waf-1 expression, and cellular growth by Pnck is independent of Pnck-induced ligand-independent EGFR degradation.
authors.affiliation: Dept. of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3970 Reservoir Road NW, Washington, DC 20057, USA. tbd@georgetown.edu
authors.forename: Tushar B
authors.initials: TB
authors.lastname: Deb
authors.forename: Annie H
authors.initials: AH
authors.lastname: Zuo
authors.forename: Youhong
authors.initials: Y
authors.lastname: Wang
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Barndt
authors.forename: Amrita K
authors.initials: AK
authors.lastname: Cheema
authors.forename: Surojeet
authors.initials: S
authors.lastname: Sengupta
authors.forename: Christine M
authors.initials: CM
authors.lastname: Coticchia
authors.forename: Michael D
authors.initials: MD
authors.lastname: Johnson
mesh_terms: D016227:Benzoquinones / Q000494:pharmacology; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1 / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D057809:HEK293 Cells; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
chemical_list: D016227:Benzoquinones; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D013912:Threonine; D012694:Serine; C512478:EGFR protein, human; D066246:ErbB Receptors; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; C517606:PNCK protein, human; D020928:Mitogen-Activated Protein Kinases; D046988:Proteasome Endopeptidase Complex; C001277:geldanamycin