RAF-1 kinase inhibitor protein-mediated cholecystokinin-2 receptor desensitization and extracellular signal-regulated kinase activation

Date

2009-06-29

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Abstract

Raf-1 kinase inhibitor protein (RKIP) is initially known as a suppressor for Raf-1-mediated ERK activation. Moreover, recent findings indicate that RKIP also has a role in G-protein-coupled receptor (GPCR) desensitization. Protein kinase C (PKC)-mediated phosphorylation at Serine 153 (S153) on RKIP switches RKIP association from Raf-1 to GPCR kinase-2 (GRK2) for inhibiting GRK2-mediated G-protein-coupled receptor (GPCR) desensitization. As a member of the GPCR superfamily, Cholecystokinin-2 receptor (CCK2R) is a physiological receptor for gastrin (G17) and activates extracellular signal-regulated kinase (ERK) via the PKC activity. The inhibition of classical PKCs (cPKC, PKC-α,-β, and -γ) by Gö6976 indicated the augment in ERK activation compared to vehicle control, suggesting cPKC’s involvement in CCK2R desensitization. CCK2R-mediated ERK activation was significantly decreased when PKC-δ was selectively silenced by siRNAs, indicating that PKC-δ, a member of the novel PKC family, mediates CCK2R-induced ERK activation. Furthermore, the data for CCK2R desensitization showed that inhibited cPKC activity by Gö6976 facilitated CCK2R desensitization. However, the silencing for PKC-α,-β, or –δ by siRNAs indicated that each knockdown of PKC isozymes attenuated CCK2R desensitization. The PKC involvement in CCK2R desensitization and ERK activation also suggested a potential role of RKIP in regulation of CCK2R activity. By either silencing or overexpressing RKIPs, I proved that RKIP acts as a suppressor for CCK2R desensitization, and the phosphorylation at S153 on RKIP plays a crucial role for inhibiting desensitization. The RKIP-mediated inhibition of CCK2R desensitization also resulted in augmentation of receptor-induced ERK activation, and this finding indicates that RKIP acts as a modulator for CCK2R-mediated signaling. The mechanism for RKIP-mediated receptor desensitization was investigated by co-immunoprecipitation of GRK2 with RKIP. The data indicated that RKIP strongly associated onto GRK2 when PKC was activated by phorbol 12-myristate 13-acetate (PMA) treatment, but either G17 stimulation or Gö6976 did not affect on the association. It suggests that PMA-sensitive PKC isozymes are responsible for RKIP phosphorylation; however, CCK2R-mediated PKC isozymes are not involved in RKIP phosphorylation directly, rather PKC activation by other cellular mechanisms mediate RKIP phosphorylation resulting in GRK2 association. Therefore, I conclude that RKIP mediates CCK2R desensitization and ERK activation through PKC activation.

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Keywords

RKIP, PKC, GRK2, GPCR desensitization, CCK2R

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