Luteinizing hormone receptor activation in ovarian granulosa cells promotes protein kinase A-dependent dephosphorylation of microtubule-associated protein 2D
Luteinizing hormone receptor activation in ovarian granulosa cells promotes protein kinase A-dependent dephosphorylation of microtubule-associated protein 2D
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DOI：
10.1210/me.2007-0457
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发表时间：
2008-07-01
影响因子：
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通讯作者：
Hunzicker-Dunn, Mary
中科院分区：
文献类型：
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作者：
Flynn, Maxfield P.;Maizels, Evelyn T.;Hunzicker-Dunn, Mary
LH诱导排卵和排卵前卵泡黄素化的作用主要通过激活颗粒细胞中cAMP依赖性蛋白激酶（PKA）介导。PKA活性通过A激酶锚定蛋白（AKAP）靶向许多细胞中的特定位置。我们以前表明，FSH诱导表达微管相关蛋白（MAP）2D，一个80 kDa的AKAP，在大鼠颗粒细胞，MAP 2D共免疫沉淀与PKA调节亚基在这些细胞。在这里，我们报告了一个快速和有针对性的去磷酸化MAP 2D在Thr 256/Thr 259治疗后，与人绒毛膜促性腺激素，LH受体激动剂。该事件通过用毛喉素或cAMP类似物处理来模拟，并被PKA抑制剂肉豆蔻酰化-PKI阻断，表明cAMP和PKA信号传导在颗粒细胞MAP 2D的磷酸化调节中的作用。此外，我们发现，Thr 256/Thr 259去磷酸化被蛋白磷酸酶2A（PP 2A）抑制剂冈田酸阻断，并通过免疫共沉淀和微囊藻毒素-琼脂糖下拉证明MAP 2D和PP 2A之间的相互作用。我们还表明，MAP 2D与糖原合成酶激酶（GSK）3 β相互作用，并在基础状态下通过该激酶在Thr 256/Thr 259处磷酸化。在用人绒毛膜促性腺激素处理后，观察到GSK 3 β在Ser 9和PP 2A B56 δ亚基在Ser 566的磷酸化增加，似乎分别导致LH受体介导的GSK 3 β抑制和PP 2A激活。综上所述，这些结果表明，AKAP MAP 2D的磷酸化状态是由LH受体介导的激酶和磷酸酶活性调节通过PKA急性调节。
The actions of LH to induce ovulation and luteinization of preovulatory follicles are mediated principally by activation of cAMP-dependent protein kinase (PKA) in granulosa cells. PKA activity is targeted to specific locations in many cells by A kinase-anchoring proteins (AKAPs). We previously showed that FSH induces expression of microtubule-associated protein (MAP) 2D, an 80-kDa AKAP, in rat granulosa cells, and that MAP2D coimmunoprecipitates with PKA-regulatory subunits in these cells. Here we report a rapid and targeted dephosphorylation of MAP2D at Thr256/Thr259 after treatment with human chorionic gonadotropin, an LH receptor agonist. This event is mimicked by treatment with forskolin or a cAMP analog and is blocked by the PKA inhibitor myristoylated-PKI, indicating a role for cAMP and PKA signaling in phosphoregulation of granulosa cell MAP2D. Furthermore, we show that Thr256/Thr259 dephosphorylation is blocked by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and demonstrate interactions between MAP2D and PP2A by coimmunoprecipitation and microcystin-agarose pulldown. We also show that MAP2D interacts with glycogen synthase kinase (GSK) 3 beta and is phosphorylated at Thr256/Thr259 by this kinase in the basal state. Increased phosphorylation of GSK3 beta at Ser9 and the PP2A B56 delta subunit at Ser566 is observed after treatment with human chorionic gonadotropin and appears to result in LH receptor-mediated inhibition of GSK3 beta and activation of PP2A, respectively. Taken together, these results show that the phosphorylation status of the AKAP MAP2D is acutely regulated by LH receptor-mediated modulation of kinase and phosphatase activities via PKA.