Conserved domains subserve novel mechanisms and functions in DKF-1, a Caenorhabditis elegans protein kinase D.
Protein kinase D (PKD) isoforms are effectors in signaling pathways controlled by diacylglycerol. PKDs contain conserved diacylglycerol binding (C1a, C1b), pleckstrin homology (PH), and Ser/Thr kinase domains. However, the properties of conserved domains may vary within the context of distinct PKD polypeptides. Such functional/structural malleability (plasticity) was explored by studying Caenorhabditis elegans D kinase family-1 (DKF-1), a PKD that governs locomotion in vivo. Phorbol ester binding with C1b alone activates classical PKDs by relieving C1-mediated inhibition. In contrast, C1a avidly ligated phorbol 12-myristate 13-acetate (PMA) and anchored DKF-1 at the plasma membrane. C1b bound PMA (moderate affinity) and cooperated with C1a in targeting DKF-1 to membranes. Mutations at a "Pro(11)" position in C1 domains were inactivating; kinase activity was minimal at PMA concentrations that stimulated wild type DKF-1 approximately 10-fold. DKF-1 mutants exhibited unchanged, maximum kinase activity after cells were incubated with high PMA concentrations. Titration in situ revealed that translocation and activation of wild type and mutant DKF-1 were tightly and quantitatively linked at all PMA concentrations. Thus, C1 domains positively regulated phosphotransferase activity by docking DKF-1 with pools of activating lipid. A PH domain inhibits kinase activity in classical PKDs. The DKF-1 PH module neither inhibited catalytic activity nor bound phosphoinositides. Consequently, the PH module is an obligatory, positive regulator of DKF-1 activity that is compromised by mutation of Lys(298) or Trp(396). Phosphorylation of Thr(588) switched on DKF-1 kinase activity. Persistent phosphorylation of Thr(588) (activation loop) promoted ubiquitinylation and proteasome-mediated degradation of DKF-1. Each DKF-1 domain displayed novel properties indicative of functional malleability (plasticity).
Feng
Hui
H
Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Ren
Min
M
Rubin
Charles S
CS
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D001839:Bombesin / Q000494:pharmacology; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D017124:Conserved Sequence; D006224:Cricetinae; D015536:Down-Regulation / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018377:Neurotransmitter Agents / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D025801:Ubiquitin / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D008565:Membrane Proteins; D018377:Neurotransmitter Agents; D025801:Ubiquitin; C089212:protein kinase D; D011493:Protein Kinase C; D001839:Bombesin