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Regulation of BAD by cAMP-dependent protein kinase is mediated via phosphorylation of a novel site, Ser155.

ABSTRACT: The interaction of BAD (Bcl-2/Bcl-X(L)-antagonist, causing cell death) with Bcl-2/Bcl-X(L) is thought to neutralize the anti-apoptotic effects of the latter proteins, and may represent one of the mechanisms by which BAD promotes apoptosis. A variety of survival signals are reported to induce the phosphorylation of BAD at Ser(112) or Ser(136), triggering its dissociation from Bcl-2/Bcl-X(L). Ser(136) is thought to be phosphorylated by protein kinase B (PKB, also called Akt), which is activated when cells are exposed to agonists that stimulate phosphatidylinositol 3-kinase (PI3K). In contrast, Ser(112) is reported to be phosphorylated by mitogen-activated protein (MAP) kinase-activated protein kinase-1 (MAPKAP-K1, also called RSK) and by cAMP-dependent protein kinase (PKA). Here we identify Ser(155) as a third phosphorylation site on BAD. We find that Ser(155) is phosphorylated preferentially by PKA in vitro and is the only residue in BAD that becomes phosphorylated when cells are exposed to cAMP-elevating agents. The phosphorylation of BAD at Ser(155) prevents it from binding to Bcl-X(L) and promotes its interaction with 14-3-3 proteins. We also provide further evidence that MAPKAP-K1 mediates the phosphorylation of Ser(112) in response to agonists that activate the classical MAP kinase pathway. However insulin-like growth factor 1, a potent activator of PI3K and PKB does not increase the phosphorylation of Ser(136) in BAD-transfected HEK-293 cells, and nor is the basal level of Ser(136) phosphorylation suppressed by inhibitors of PI3K.

SUBMITTER: Lizcano JM

PROVIDER: S-EPMC1221178 | biostudies-other | 2000 Jul

REPOSITORIES: biostudies-other

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Project description:Objective:Neuropathic pain (NP) is a debilitating condition caused by nervous system injury and chronic diseases. LncRNA H19 is upregulated in many human diseases, including NP. Cyclin-dependent kinase 5 (CDK5) aggressively worsens inflammatory action and nerve damage to cause severe NP. Phosphorylated cAMP response element binding protein (CREB) is detrimental to nerves and promotes NP progression. Herein, aim of our study was to assess the mechanism of lncRNA H19. Methods:The NP rat model was established using chronic constriction injury (CCI). Paw withdrawal threshold (PWT) tests and paw withdrawal latency (PWL) tests were performed. Then, small interfering (si)RNA against H19 was intrathecally injected into rats to suppress H19 expression. Schwann cells were isolated from NP rats and transfected with siRNA-H19 or a lentivirus (LV)-based vector expressing H19. Inflammatory factors and glial fibrillary acidic protein (GFAP) were detected. Western blot analysis was conducted to detect CDK5/p35 and p-CREB expression. Finally, H19, CDK5 and CREB phosphorylation were tested with the combination of the CDK5 inhibitor roscovitine and transfection of LV-H19 and siRNA-H19. Finally, we investigated the binding relationships between H19 and miR-196a-5p and between miR-196a-5p and CDK5 and detected the mRNA expression of miR-196a-5p and CDK5 in rats with H19 knockdown and in Schwann cells with H19 knockdown. Results:Highly expressed H19, CDK5/p-35 and p-CREB were observed in NP rats, accompanied by obviously decreased PWT and PWL, upregulated inflammatory factors and GFAP levels, and reduced 5-HT2A and GABAB2 expression. siRNA-H19 restored NP-related indexes and downregulated CDK5/p35 and p-CREB phosphorylation. siRNA-H19, together with the CDK5 inhibitor roscovitine, reduced CDK5 and p-CREB expression in Schwann cells isolated from NP rats. Binding sites between H19 and miR-196a-5p and between miR-196a-5p and CDK5 were identified. Silencing H19 upregulated miR-196a-5p expression and downregulated CDK5 levels. Conclusion:Our study demonstrated that silencing H19 inhibited NP by suppressing CDK5/p35 and p-CREB phosphorylation via the miR-196a-5p/CDK5 axis, which may provide new insight into NP treatment.

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Regulation of BAD by cAMP-dependent protein kinase is mediated via phosphorylation of a novel site, Ser155.

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