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 ...