title: Regulatory domain determinants that control PKD1 activity.
abstract: The canonical pathway for protein kinase D1 (PKD1) activation by growth factor receptors involves diacylglycerol binding to the C1 domain and protein kinase C-dependent phosphorylation at the activation loop. PKD1 then autophosphorylates at Ser(916), a modification frequently used as a surrogate marker of PKD1 activity. PKD1 also is cleaved by caspase-3 at a site in the C1-PH interdomain during apoptosis; the functional consequences of this cleavage event remain uncertain. This study shows that PKD1-Δ1-321 (an N-terminal deletion mutant lacking the C1 domain and flanking sequence that models the catalytic fragment that accumulates during apoptosis) and PKD1-CD (the isolated catalytic domain) display high basal Ser(916) autocatalytic activity and robust activity toward CREBtide (a peptide substrate) but little to no activation loop autophosphorylation and no associated activity toward protein substrates, such as cAMP-response element binding protein and cardiac troponin I. In contrast, PKD1-ΔPH (a PH domain deletion mutant) is recovered as a constitutively active enzyme, with high basal autocatalytic activity and high basal activity toward peptide and protein substrates. These results indicate that individual regions in the regulatory domain act in a distinct manner to control PKD1 activity. Finally, cell-based studies show that PKD1-Δ1-321 does not substitute for WT-PKD1 as an in vivo activator of cAMP-response element binding protein and ERK phosphorylation. Proteolytic events that remove the C1 domain (but not the autoinhibitory PH domain) limit maximal PKD1 activity toward physiologically relevant protein substrates and lead to a defect in PKD1-dependent cellular responses.
authors.affiliation: Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
authors.forename: Vitalyi O
authors.initials: VO
authors.lastname: Rybin
authors.forename: Jianfen
authors.initials: J
authors.lastname: Guo
authors.forename: Erin
authors.initials: E
authors.lastname: Harleton
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Susan F
authors.initials: SF
authors.lastname: Steinberg
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D050882:CREB-Binding Protein / Q000378:metabolism; D020134:Catalytic Domain; D004789:Enzyme Activation / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000201:enzymology* / Q000473:pathology; D041681:NIH 3T3 Cells; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D050396:TRPP Cation Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019210:Troponin I / Q000378:metabolism; D020257:Ventricular Remodeling / Q000502:physiology
chemical_list: D050882:CREB-Binding Protein; D048049:Extracellular Signal-Regulated MAP Kinases; D050396:TRPP Cation Channels; D019210:Troponin I