Regulation of death-associated protein kinase. Stabilization by HSP90 heterocomplexes.
Death-associated protein kinase (DAPK) has been found associated with HSP90, and inhibition of HSP90 with 17-alkylamino-17-demethoxygeldanamycin reduced expression of DAPK. These results were extended to determine whether the degradation of DAPK in the absence of HSP90 activity is dependent on the ubiquitin-proteasome pathway. Our results show that treatment of cells with geldanamycin (GA) leads to degradation of DAPK, and this degradation is attenuated by the proteasome inhibitor, lactacystin. GA-induced DAPK degradation is also dependent on phosphorylation of DAPK at Ser(308), and the cellular levels of phospho(Ser(308))-DAPK dramatically increase in response to GA treatment. Expression of two distinct ubiquitin E3 ligases, carboxyl terminus of HSC70-interacting protein (CHIP) or DIP1/Mib1, enhanced DAPK degradation, and conversely, short interfering RNA depletion of either CHIP or DIP1/Mib1 attenuated DAPK degradation. In vitro ubiquitination assays confirmed that DAPK is targeted for ubiquitination by both CHIP and DIP. Consistent with these results, DAPK is found in two distinct immune complexes, one containing HSP90 and CHIP and a second complex containing only DIP1/Mib. Collectively, these results indicate that strict modulation of DAPK activities is critical for regulation of apoptosis and cellular homeostasis.
Zhang
Liguo
L
Department of Cellular and Integrated Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Nephew
Kenneth P
KP
Gallagher
Patricia J
PJ
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis*; D016227:Benzoquinones / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000096:biosynthesis*; D002384:Catalysis; D064550:Death-Associated Protein Kinases; D015971:Gene Expression Regulation, Enzymologic; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D016227:Benzoquinones; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D011994:Recombinant Proteins; D025801:Ubiquitin; D012694:Serine; C112765:tanespimycin; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D046988:Proteasome Endopeptidase Complex