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 "Pro 11 " position in C1 domains were inactivating; kinase activity was minimal at PMA concentrations that stimulated wild type DKF-1 ϳ10-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). The protein kinase D (PKD)2 family of Ser/Thr protein kinases (PKD, PKD2, and PKD3) mediates transmission and targeting of signals carried by the second messenger, diacylglycerol (DAG) (1-3). PKD activation is correlated with enhanced NFB-mediated gene transcription, oxidative stress responses, changes in cell adhesion, Golgi vesicle generation and trafficking, apoptosis, and T-cell activation and secretion. The functions of PKDs are governed by four conserved structural modules (4 -7). Tandem C1a and C1b domains, located near the PKD N terminus, bind DAG or phorbol esters (e.g. phorbol 12-myristate 13-acetate (PMA)) that mimic DAG. A central PH domain inhibits kinase activity (8) but also ligates PKD-activating proteins (9 -11). Catalytic activity of the C-terminal kinase domain is expressed when two Ser hydroxyl groups in the activation loop undergo trans phosphorylation (12, 13). PMA/DAG-activated protein kinase C (PKC) isoforms phosphorylate the PKD activation loop (1-3). Thus, PKDs are PKC effectors in signaling pathways governed by hormones or growth factors that activate phospholipases C ␤ , C ␥ , or C . PKDs disseminate and diversify DAG/PKC s...

Properties, Regulation, and in Vivo Functions of a Novel Protein Kinase D

“…Studies on DKF-1, a C. elegans PKD, confirmed the value of the model system (17,27). DKF-1 has the substrate specificity and structural hallmarks of a typical PKD.…”

“…Preparation of Cytosol and Total Membrane Proteins in Translocation Assays-Cells were disrupted in hypotonic detergent-free buffer, as stated previously (17,31). Cytosol (supernatant) and total membranes (pellet) were isolated by centrifugation at 120,000 ϫ g. Volumes of cytosol and pellet fractions were adjusted so that equal aliquots represent equal numbers of starting cells (normalization to protein could also be used; cytosol and pellet each contained 50% of total protein).…”

“…Duplicate batches of WT and mutant animals were maintained at 20°C and assayed in parallel. Animals were mounted, and subcellular location of DAF-16-GFP was determined by fluorescence microscopy as described previously (17,27).…”

“…Other Experimental Procedures-Detailed descriptions of PCR amplification and analysis of a partially deleted gene, generation and analysis of transgenic animals, confocal immunofluorescence microscopy, protein determination, and deletion and site-directed mutagenesis are provided in recent papers (17,27).…”

“…One puzzle is that homologous regulatory domains control catalytic activity via dissimilar mechanisms in different PKDs (5,17). For example, the PKD1 PH domain inhibits catalytic activity (18), whereas PH domain mutations in a Caenorhabditis elegans PKD (named DKF-1, for D kinase family 1) extinguish kinase activity (17). Physiological roles of PKDs have been inferred from studies on transfected cells, but it is not known if roles assigned to PKDs are major or minor functions of D kinases in normal differentiated cells.…”