PKD is a kinase of Vps34 that mediates ROS-induced autophagy downstream of DAPk

Autophagy, a process in which cellular components are engulfed and degraded within double-membrane vesicles termed autophagosomes, has an important role in the response to oxidative damage. Here we identify a novel cascade of phosphorylation events, involving a network of protein and lipid kinases, as crucial components of the signaling pathways that regulate the induction of autophagy under oxidative stress. Our findings show that both the tumor-suppressor death-associated protein kinase (DAPk) and protein kinase D (PKD), which we previously showed to be phosphorylated and consequently activated by DAPk, mediate the induction of autophagy in response to oxidative damage. Furthermore, we map the position of PKD within the autophagic network to Vps34, a lipid kinase whose function is indispensable for autophagy, and demonstrate that PKD is found in the same molecular complex with Vps34. PKD phosphorylates Vps34, leading to activation of Vps34, phosphatydilinositol-3-phosphate (PI(3)P) formation, and autophagosome formation. Consistent with its identification as a novel inducer of the autophagic machinery, we show that PKD is recruited to LC3-positive autophagosomes, where it localizes specifically to the autophagosomal membranes. Taken together, our results describe PKD as a novel Vps34 kinase that functions as an effecter of autophagy under oxidative stress

Sphingomyelin Synthases Regulate Protein Trafficking and Secretion

Sphingomyelin synthases (SMS1 and 2) represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG). SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein, protein kinase D (PKD), to the Golgi. Since PKD recruitment to the Golgi has been implicated in cellular secretion through the trans golgi network (TGN), the effect of down-regulation of SMSs on TGN-to-plasma membrane trafficking was studied. Down regulation of either SMS1 or SMS2 significantly retarded trafficking of the reporter protein vesicular stomatitis virus G protein tagged with GFP (VSVG-GFP) from the TGN to the cell surface. Inhibition of SMSs also induced tubular protrusions from the trans Golgi network reminiscent of inhibited TGN membrane fission. Since a recent study demonstrated the requirement of PKD activity for insulin secretion in beta cells, we tested the function of SMS in this model. Inhibition of SMS significantly reduced insulin secretion in rat INS-1 cells. Taken together these results provide the first direct evidence that both enzymes (SMS1 and 2) are capable of regulating TGN-mediated protein trafficking and secretion, functions that are compatible with PKD being a down-stream target for SMSs in the Golgi

Protein kinase D interacts with neuronal nitric oxide synthase and phosphorylates the activatory residue serine1412

Neuronal Nitric Oxide Synthase (nNOS) is the biosynthetic enzyme responsible for nitric oxide (·NO) production in muscles and in the nervous system. This constitutive enzyme, unlike its endothelial and inducible counterparts, presents an N-terminal PDZ domain known to display a preference for PDZ-binding motifs bearing acidic residues at -2 position. In a previous work, we discovered that the C-terminal end of two members of protein kinase D family (PKD1 and PKD2) constitutes a PDZ-ligand. PKD1 has been shown to regulate multiple cellular processes and, when activated, becomes autophosphorylated at Ser 916, a residue located at -2 position of its PDZ-binding motif. Since nNOS and PKD are spatially enriched in postsynaptic densities and dendrites, the main objective of our study was to determine whether PKD1 activation could result in a direct interaction with nNOS through their respective PDZ-ligand and PDZ domain, and to analyze the functional consequences of this interaction. Herein we demonstrate that PKD1 associates with nNOS in neurons and in transfected cells, and that kinase activation enhances PKD1-nNOS co-immunoprecipitation and subcellular colocalization. However, transfection of mammalian cells with PKD1 mutants and yeast two hybrid assays showed that the association of these two enzymes does not depend on PKD1 PDZ-ligand but its pleckstrin homology domain. Furthermore, this domain was able to pull-down nNOS from brain extracts and bind to purified nNOS, indicating that it mediates a direct PKD1-nNOS interaction. In addition, using mass spectrometry we demonstrate that PKD1 specifically phosphorylates nNOS in the activatory residue Ser 1412, and that this phosphorylation increases nNOS activity and ·NO production in living cells. In conclusion, these novel findings reveal a crucial role of PKD1 in the regulation of nNOS activation and synthesis of ·NO, a mediator involved in physiological neuronal signaling or neurotoxicity under pathological conditions such as ischemic stroke or neurodegeneration.This work was supported by the Ministerio de Economía y Competitividad [SAF2011-26233 to T.I., BFU2009-10442 and BFU2012-37934 to I.R-C.]; Comunidad de Madrid [S2010/BMD-2331-Neurodegmodels-CM to T.I.]; and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas – CIBERNED, Instituto de Salud Carlos III, to T.I. Postdoctoral fellows L.S-R. and L.G-G. have been funded by research contracts from CIBERNED; Clara Aicart-Ramos is a recipient of a FPU predoctoral fellowship from Ministerio de Economía y Competitividad.Peer Reviewe

Regulation of Oxysterol-binding Protein Golgi Localization through Protein Kinase D–mediated Phosphorylation

Protein kinase D (PKD) is a critical regulator of Golgi structure and function. Biochemical evidence is presented that demonstrates the oxysterol-binding protein OSBP as a novel PKD substrate. Phosphorylation inhibits OSBP Golgi localization, impairs CERT Golgi localization, and promotes Golgi fragmentation