title: MARCKS dephosphorylation is involved in bradykinin-induced neurite outgrowth in neuroblastoma SH-SY5Y cells.
abstract: Bradykinin (BK) plays a major role in producing peripheral sensitization in response to peripheral inflammation and in pain transmission in the central nerve system (CNS). Because BK activates protein kinase C (PKC) through phospholipase C (PLC)-β and myristoylated alanine-rich C kinase substrate (MARCKS) has been found to be a substrate of PKC, we explored the possibility that BK could induce MARCKS phosphorylation and regulate its function. BK stimulation induced transient MARCKS phosphorylation on Ser159 with a peak at 1  min in human neuroblastoma SH-SY5Y cells. By contrast, PKC activation by the phorbol ester phorbol 12,13-dibutyrate (PDBu) elicited MARCKS phosphorylation which lasted more than 10  min. Western blotting analyses and glutathione S-transferase (GST) pull-down analyses showed that the phosphorylation by BK was the result of activation of the PKC-dependent RhoA/Rho-associated coiled-coil kinase (ROCK) pathway. Protein phosphatase (PP) 2A inhibitors calyculin A and fostriecin inhibited the dephosphorylation of MARCKS after BK-induced phosphorylation. Moreover, immunoprecipitation analyses showed that PP2A interacts with MARCKS. These results indicated that PP2A is the dominant PP of MARCKS after BK stimulation. We established SH-SY5Y cell lines expressing wild-type MARCKS and unphosphorylatable MARCKS, and cell morphology changes after cell stimulation were studied. PDBu induced lamellipodia formation on the neuroblastoma cell line SH-SY5Y and the morphology was sustained, whereas BK induced neurite outgrowth of the cells via lamellipodia-like actin accumulation that depended on transient MARCKS phosphorylation. Thus these findings show a novel BK signal cascade-that is, BK promotes neurite outgrowth through transient MARCKS phosphorylation involving the PKC-dependent RhoA/ROCK pathway and PP2A in a neuroblastoma cell line.
authors: Laboratory of Pharmacology, School of Pharmacy, Kitasato University, Minato-ku, Tokyo, Japan. tanabea@pharm.kitasato-u.ac.jp
authors: Atsuhiro
authors: A
authors: Tanabe
authors: Mitsuya
authors: M
authors: Shiraishi
authors: Manabu
authors: M
authors: Negishi
authors: Naoaki
authors: N
authors: Saito
authors: Mitsuo
authors: M
authors: Tanabe
authors: Yasuharu
authors: Y
authors: Sasaki
mesh_terms: D000199:Actins / Q000378:metabolism; D001920:Bradykinin / Q000494:pharmacology*; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D016501:Neurites / Q000502:physiology*; D009447:Neuroblastoma / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D011554:Pseudopodia / Q000502:physiology; D043782:Receptor, Bradykinin B2 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D000199:Actins; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D043782:Receptor, Bradykinin B2; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D001920:Bradykinin