title: Phosphorylation of pleckstrin increases proinflammatory cytokine secretion by mononuclear phagocytes in diabetes mellitus.
abstract: The protein kinase C (PKC) family of intracellular enzymes plays a crucial role in signal transduction for a variety of cellular responses of mononuclear phagocytes including phagocytosis, oxidative burst, and secretion. Alterations in the activation pathways of PKC in a variety of cell types have been implicated in the pathogenesis of the complications of diabetes. In this study, we investigated the consequences of PKC activation by evaluating endogenous phosphorylation of PKC substrates with a phosphospecific PKC substrate Ab (pPKC(s)). Phosphorylation of a 40-kDa protein was significantly increased in mononuclear phagocytes from diabetics. Phosphorylation of this protein is downstream of PKC activation and its phosphorylated form was found to be associated with the membrane. Mass spectrometry analysis, immunoprecipitation, and immunoblotting experiments revealed that this 40-kDa protein is pleckstrin. We then investigated the phosphorylation and translocation of pleckstrin in response to the activation of receptor for advanced glycation end products (RAGE). The results suggest that pleckstrin is involved in RAGE signaling and advanced glycation end product (AGE)-elicited mononuclear phagocyte dysfunction. Suppression of pleckstrin expression with RNA interference silencing revealed that phosphorylation of pleckstrin is an important intermediate in the secretion and activation pathways of proinflammatory cytokines (TNF-alpha and IL-1beta) induced by RAGE activation. In summary, this study demonstrates that phosphorylation of pleckstrin is up-regulated in diabetic mononuclear phagocytes. The phosphorylation is in part due to the activation of PKC through RAGE binding, and pleckstrin is a critical molecule for proinflammatory cytokine secretion in response to elevated AGE in diabetes.
authors.affiliation: Department of Periodontology and Oral Biology, Goldman School of Dental Medicine, Boston University, Boston, MA 02118, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Ding
authors.forename: Alpdogan
authors.initials: A
authors.lastname: Kantarci
authors.forename: John A
authors.initials: JA
authors.lastname: Badwey
authors.forename: Hatice
authors.initials: H
authors.lastname: Hasturk
authors.forename: Alan
authors.initials: A
authors.lastname: Malabanan
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Van Dyke
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000378:metabolism* / Q000502:physiology; D045744:Cell Line, Tumor; D016207:Cytokines / Q000378:metabolism* / Q000502:physiology; D003920:Diabetes Mellitus / Q000276:immunology* / Q000378:metabolism / Q000473:pathology*; D004789:Enzyme Activation / Q000276:immunology; D005260:Female; D017127:Glycation End Products, Advanced / Q000378:metabolism / Q000502:physiology; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism* / Q000502:physiology; D008264:Macrophages / Q000201:enzymology / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009000:Monocytes / Q000201:enzymology / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D010750:Phosphoproteins / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000037:antagonists & inhibitors / Q000502:physiology; D015398:Signal Transduction / Q000276:immunology; D015854:Up-Regulation / Q000276:immunology*
chemical_list: D001798:Blood Proteins; D016207:Cytokines; D017127:Glycation End Products, Advanced; D018836:Inflammation Mediators; D010750:Phosphoproteins; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; C039077:platelet protein P47; D011493:Protein Kinase C