title: PKCβII acts downstream of chemoattractant receptors and mTORC2 to regulate cAMP production and myosin II activity in neutrophils.
abstract: Chemotaxis is a process by which cells polarize and move up a chemical gradient through the spatiotemporal regulation of actin assembly and actomyosin contractility, which ultimately control front protrusions and back retractions. We previously demonstrated that in neutrophils, mammalian target of rapamycin complex 2 (mTORC2) is required for chemoattractant-mediated activation of adenylyl cyclase 9 (AC9), which converts ATP into cAMP and regulates back contraction through MyoII phosphorylation. Here we study the mechanism by which mTORC2 regulates neutrophil chemotaxis and AC9 activity. We show that inhibition of protein kinase CβII (PKCβII) by CPG53353 or short hairpin RNA knockdown severely inhibits chemoattractant-induced cAMP synthesis and chemotaxis in neutrophils. Remarkably, PKCβII-inhibited cells exhibit specific and severe tail retraction defects. In response to chemoattractant stimulation, phosphorylated PKCβII, but not PKCα, is transiently translocated to the plasma membrane, where it phosphorylates and activates AC9. mTORC2-mediated PKCβII phosphorylation on its turn motif, but not its hydrophobic motif, is required for membrane translocation of PKCβII. Inhibition of mTORC2 activity by Rictor knockdown not only dramatically decreases PKCβII activity, but it also strongly inhibits membrane translocation of PKCβII. Together our findings show that PKCβII is specifically required for mTORC2-dependent AC9 activation and back retraction during neutrophil chemotaxis.
authors.affiliation: Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Lunhua
authors.initials: L
authors.lastname: Liu
authors.forename: Derek
authors.initials: D
authors.lastname: Gritz
authors.forename: Carole A
authors.initials: CA
authors.lastname: Parent
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002634:Chemotaxis, Leukocyte; D000242:Cyclic AMP / Q000096:biosynthesis*; D004789:Enzyme Activation; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D000076225:Mechanistic Target of Rapamycin Complex 2; D046912:Multiprotein Complexes / Q000378:metabolism*; D024462:Myosin Type II; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology* / Q000502:physiology; D010766:Phosphorylation; D010797:Phthalimides / Q000494:pharmacology; D064546:Protein Kinase C beta / Q000037:antagonists & inhibitors / Q000502:physiology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D044042:Receptors, Formyl Peptide / Q000378:metabolism*; D015290:Second Messenger Systems; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D010797:Phthalimides; D047428:Protein Kinase Inhibitors; D044042:Receptors, Formyl Peptide; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000242:Cyclic AMP; D000076225:Mechanistic Target of Rapamycin Complex 2; D058570:TOR Serine-Threonine Kinases; C579054:PRKCB protein, human; D064546:Protein Kinase C beta; D024462:Myosin Type II; D000262:Adenylyl Cyclases; C098874:4,5-dianilinophthalimide