Conserved domains subserve novel mechanisms and functions in DKF-1, a Caenorhabditis elegans protein kinase D

Abstract

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 "Pro11" position in C1 domains were inactivating; kinase activity was minimal at PMA concentrations that stimulated wild type DKF-1 ∼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 Lys298 or Trp396. Phosphorylation of Thr588 switched on DKF-1 kinase activity. Persistent phosphorylation of Thr588 (activation loop) promoted ubiquitinylation and proteasome-mediated degradation of DKF-1. Each DKF-1 domain displayed novel properties indicative of functional malleability (plasticity). © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.