Phosphorylation of GRK2 by protein kinase C abolishes its inhibition by calmodulin
Phosphorylation of GRK2 by protein kinase C abolishes its inhibition by calmodulin
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DOI：
10.1074/jbc.m008773200
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发表时间：
2001-01-19
影响因子：
4.8
通讯作者：
Lohse, MJ
中科院分区：
文献类型：
--
作者：
Krasel, C;Dammeier, S;Lohse, MJ
G蛋白偶联受体激酶（GRKs）是G蛋白偶联受体功能的重要调节因子。该家族的两个成员GRK 2和GRK 5 L已被证明是蛋白激酶C（PKC）的底物。而PRC介导的磷酸化导致GRK 5的抑制，它可能通过增加对含受体膜的亲和力来增加GRK 2对其底物的活性。我们在这里表明，这种活性的增加可能是由钙调素缓解紧张性抑制GRK 2。在体外，GRK 2优先被PKC亚型α、γ和δ磷酸化。PHC α-磷酸化GRK 2的二维肽图谱显示单一磷酸化位点，其通过HPLC-MS鉴定为丝氨酸29。GRK 2的S29 A突变体在体外不被PKC磷酸化，并且当转染到人胚肾（HEK）293细胞中时不显示佛波酯刺激的磷酸化。丝氨酸29位于GRK 2的钙调素结合区，钙调素与GRK 2的结合导致激酶活性的抑制。当GRK 2被PHC磷酸化时，这种抑制作用在体外几乎完全消除。这些数据表明，钙调素可能是GRK 2的抑制剂，其作用可以被PKC介导的GRK 2磷酸化所消除。
G-protein-coupled receptor kinases (GRKs) are important regulators of G-protein-coupled receptor function. Two members of this family L, GRK2 and GRK5 L, have been shown to be substrates for protein kinase C (PKC). Whereas PRC-mediated phosphorylation results in inhibition of GRK5, it increases the activity of GRK2 toward its substrates probably through increased affinity for receptor-containing membranes. We show here that this increase in activity may be caused by relieving a tonic inhibition of GRK2 by calmodulin. In vitro, GRK2 was preferentially phosphorylated by PKC isoforms alpha, gamma, and delta, Two-dimensional peptide mapping of PHC alpha -phosphorylated GRK2 showed a single site of phosphorylation, which was identified as serine 29 by HPLC-MS. A S29A mutant of GRK2 was not phosphorylated by PKC in vitro and showed no phorbol ester-stimulated phosphorylation when transfected into human embryonic kidney (HEK)293 cells. Serine 29 is located in the calmodulinbinding region of GRK2, and binding of calmodulin to GRK2 results in inhibition of kinase activity. This inhibition was almost completely abolished in vitro when GRK2 was phosphorylated by PHC. These data suggest that calmodulin may be an inhibitor of GRK2 whose effects can be abolished with PKC-mediated phosphorylation of GRK2.