PKDs (DKF-2A/2B) and a PKD effector (HDA-4) regulate Caenorhabditis elegans' responses to sodium and pathogens

Abstract

Protein kinase D (PKD) is a ubiquitously-expressed, target/effector for hormones and neurotransmitters that promote diacylglycerol (DAG) synthesis. This thesis addresses deficiencies in knowledge of physiological roles and in vivo regulation of PKDs. The C. elegans dkf-2 gene encodes a novel PKD, DKF-2B, in neurons that constitute chemosensory circuitry, and a previously-characterized PKD (DKF-2A) in intestinal cells. Characterization of DKF-2A/2B deficient C. elegans and mutant animals reconstituted with DKF-2A and/or 2B revealed that PKDs place a neurophysiological process, experience-dependent learning, under DAG control. DKF-2B and 2A are indispensable for Na+ (salt taste)-induced learning and behavioral plasticity. A phospholipase Cbeta4 ortholog (EGL-8) and TPA-1, a protein kinase C delta/theta homolog, regulate DKF-2A/2B in vivo. Thus, EGL-8, TPA-1 and DKF-2A/2B constitute a regulatory module that generates and transmits DAG signals in vivo. A novel role for PKDs was established: learning was linked to signal integration within a PKD-controlled binary detector system embedded in nervous tissue and the gut.;Activation of DKF-2A triggers induction of >75 immune effector proteins that protect C. elegans intestinal cells against bacterial pathogens. DKF-2A activation and pathogens also induce accumulation of HDA-4, a histone deacetylase (HDAC), and MEF-2, a transcription factor that binds HDA-4. HDACs repress genes by deacetylating histones (altering chromatin structure) and binding transcriptional activators. I tested the hypothesis that the HDA-4/MEF-2 complex is a DKF-2A target/effector that regulates innate immunity. Non-phosphorylated HDA-4 accumulated in nuclei and repressed gene transcription. Activated DKF-2A phosphorylated HDA-4. Phospho-HDA-4 dissociated from MEF-2, translocated to cytoplasm and bound 14-3-3 protein. A loss-of-function mutation in the hda-4 gene (hda-4(oy57)) rendered C. elegans super-resistant to infection by P. aeruginosa (PA14). Microarray analysis revealed HDA-4 deficiency (MEF-2 activation) promoted induction of mRNAs encoding numerous anti-microbial proteins. DKF-2A and a DKF-2A effector, PMK-1 (p38 MAP kinase), were indispensable for MEF-2 regulated pathogen resistance. Animals lacking MEF-2 (wild type or hda-4(oy57) background) were hyper-susceptible to PA14 infection/killing, indicating that MEF-2 and HDA-4 are in the same signaling pathway. Thus, a signaling module that includes DKF-2A, PMK-1 and HDA-4/MEF-2 mediates induction of innate immunity.