title: The protein kinase D1 COOH terminus: marker or regulator of enzyme activity?
abstract: Protein kinase D1 (PKD1) is a Ser/Thr kinase implicated in a wide variety of cellular responses. PKD1 activation is generally attributed to a PKC-dependent pathway that leads to phosphorylation of the activation loop at Ser(744)/Ser(748). This modification increases catalytic activity, including that toward an autophosphorylation site (Ser(916)) in a postsynaptic density-95/disks large/zonula occludens-1 (PDZ)-binding motif at the extreme COOH terminus. However, there is growing evidence that PKD1 activation can also result from a PKC-independent autocatalytic reaction at Ser(744)/Ser(748) and that certain stimuli increase in PKD1 phosphorylation at Ser(744)/S(748) without an increase in autophosphorylation at Ser(916). This study exposes a mechanism that results in a discrepancy between PKD1 COOH-terminal autocatalytic activity and activity toward other substrates. We show that PKD1 constructs harboring COOH-terminal epitope tags display high levels of in vitro activation loop autocatalytic activity and activity toward syntide-2 (a peptide substrate), but no Ser(916) autocatalytic activity. Cell-based studies show that the COOH-terminal tag, adjacent to PKD1's PDZ1-binding motif, does not grossly influence PKD1 partitioning between soluble and particulate fractions in resting cells or PKD1 translocation to the particulate fraction following treatment with PMA. However, a COOH-terminal tag that confers a high level of activation loop autocatalytic activity decreases the PKC requirement for agonist-dependent PKD1 activation in cells. The recognition that COOH-terminal tags alter PKD1's pharmacological profile is important from a technical standpoint. The altered dynamics and activation mechanisms for COOH-terminal-tagged PKD1 enzymes also could model the signaling properties of localized pools of enzyme anchored through the COOH terminus to PDZ domain-containing scaffolding proteins.
authors: Department of Pharmacology, Columbia University, New York, New York.
authors: Weihua
authors: W
authors: Qiu
authors: Department of Pharmacology, Columbia University, New York, New York.
authors: Fan
authors: F
authors: Zhang
authors: Department of Pharmacology, Columbia University, New York, New York sfs1@columbia.edu.
authors: Susan F
authors: SF
authors: Steinberg
mesh_terms: D020134:Catalytic Domain; D004789:Enzyme Activation; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D054731:PDZ Domains; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C063391:syntide-2; D012694:Serine; C089212:protein kinase D; D011493:Protein Kinase C
keywords: phosphorylation; postsynaptic density-95/disks large/zonula occludens-1-binding motif; protein kinase D