A Novel Conserved Domain Mediates Dimerization of Protein Kinase D (PKD) Isoforms

Abstract

Edited by Alex TokerProtein kinase D (PKD) isoforms are protein kinase C effectors in signaling pathways regulated by diacylglycerol. Important physiological processes (including secretion, immune responses, motility, and transcription) are placed under diacylglycerol control by the distinctive substrate specificity and subcellular distribution of PKDs. Potentially, broadly co-expressed PKD polypeptides may interact to generate homo-or heteromultimeric regulatory complexes. However, the frequency, molecular basis, regulatory significance, and physiological relevance of stable PKD-PKD interactions are largely unknown. Here, we demonstrate that mammalian PKDs 1-3 and the prototypical Caenorhabditis elegans PKD, DKF-2A, are exclusively (homo-or hetero-) dimers in cell extracts and intact cells. We discovered and characterized a novel, highly conserved N-terminal domain, comprising 92 amino acids, which mediates dimerization of PKD1, PKD2, and PKD3 monomers. A similar domain directs DKF-2A homodimerization. Dimerization occurred independently of properties of the regulatory and kinase domains of PKDs. Disruption of PKD dimerization abrogates secretion of PAUF, a protein carried in small trans-Golgi network-derived vesicles. In addition, disruption of DKF-2A homodimerization in C. elegans intestine impaired and degraded the immune defense of the intact animal against an ingested bacterial pathogen. Finally, dimerization was indispensable for the strong, dominant negative effect of catalytically inactive PKDs. Overall, the structural integrity and function of the novel dimerization domain are essential for PKDmediated regulation of a key aspect of cell physiology, secretion, and innate immunity in vivo.The membrane-embedded second messenger diacylglycerol (DAG) 2 mediates actions of many hormones and other stimuli by activating two classes of PKC isoforms (1, 2). Conventional PKCs ␣, ␤I, ␤II, and ␥ are stimulated by DAG and Ca 2ϩ acting in concert; novel PKCs ␦, ⑀, , and are activated by DAG alone. PKCs regulate many aspects of cell physiology by activating members of another family of Ser/Thr kinases, collectively named PKD (3-6). Like PKCs, PKDs have a C-terminal kinase domain that is preceded by C1a and C1b regulatory domains, which bind DAG and a pharmacological activator, phorbol 12-myristate 13-acetate (PMA). C1 domains mediate translocation of PKDs and PKCs from cytoplasm to DAG/PMA-enriched membranes. DAG/PMA-activated PKC phosphorylates the PKD activation loop (A-loop), thereby switching on catalytic activity of the co-recruited D kinase. Thus, PKDs are PKC effectors in regulatory cascades.PKDs diversify DAG/PKC signaling by acting on effectors in plasma membrane, the nucleus, cytoplasm, F-actin cytoskeleton, and cytoplasmic surfaces of mitochondria and Golgi membranes. Because PKCs and PKDs phosphorylate different substrates (3, 4, 7), D kinases place distinct effectors and physiological processes under DAG control. For instance, PKDs mediate pro-survival signaling induced by oxidative stress (8); control ...

It Takes Two to Tango: Activation of Protein Kinase D by Dimerization

“…Vertebrate PKD1 and PKD2 additionally encode a C‐terminal PDZ motif ( Figure 3 a). Consistent with a number of studies that have reported a functional requirement for dimerization of PKD, a conserved ubiquitin‐like domain (ULD, Figure 3b) that mediates dimerization was recently identified and characterized in human PKD1‐3 and Dkf‐1, a homolog of PKD in Caenorhabditis elegans . Dkf‐1, at just 720 amino acids, has one of the most compact PKD sequences, making it an invaluable template for annotating the core elements required for PKD function (Figure 3a,b).…”

“…ULD‐mediated dimerization of PKD explains the strong, dominant negative effect of overexpression of catalytically inactive PKD or truncated PKD comprising only its regulatory domains . These dominant negative effects may also extend to cross‐isoform inhibition under conditions of ectopic over‐expression, since the ULD dimerization interface is invariant across PKD1, 2, and 3 .…”

“…A dimer of PKD would be expected to translocate faster than a monomer by virtue of the avidity effect of having four C1 domains compared with two. However, size‐exclusion chromatography and sucrose gradient centrifugation of detergent‐solubilized cell lysates containing C. elegans Dkf‐2 or human PKD1/PKD2 yielded elongated dimers in each case, leading the authors to conclude that PKD is constitutively dimeric . It should be noted, though, that over‐expression may have driven dimer formation in these cells and that detergent‐solubilization could recover dimeric PKD in an active (and hence more extended) conformation.…”

“…Both autophosphorylation and trans-phosphorylation by protein kinase C (PKC) have been proposed to drive PKD activation, but a mechanistic understanding of PKD activation at the membrane has been lacking. The recent identification and structure determination of a novel dimerization domain in the N-terminus of PKD [14,15] raises many questions that will guide future efforts to understand the structure, biochemistry, and function of PKD.…”

Protein kinase D displays intrinsic Tyr autophosphorylation activity: insights into mechanism and regulation

“…They consist of a large N‐terminal domain, followed by the kinase domain. The N‐terminal regulatory region encompasses an alanine/proline rich region (AP region), a tandem C1 domain binding diacylglycerol (DAG) and phorbol esters, an oligomerization domain, and a pleckstrin homology (PH) domain 24, 25, 26, 27, 28. Deletion mutagenesis approaches have indicated that the C1 and PH domains have an autoinhibitory effect on kinase activity, which is alleviated by activation loop Ser‐738/742 phosphorylation (hPKD1 numbering) 24, 25, 26.…”