Hunter S, Koch B L, Anderson S M
University of Colorado Health Sciences Center, Department of Pathology, Denver 80262, USA.
Mol Endocrinol. 1997 Aug;11(9):1213-22. doi: 10.1210/mend.11.9.9980.
Stimulation of Nb2 cells with PRL results in the rapid phosphorylation of a 120-kDa protein identified as the adapter protein cbl on tyrosine residues. Maximal phosphorylation of cbl occurs at 20 min after PRL stimulation and declines thereafter. Stimulation with as little as 5 nM PRL resulted in the phosphorylation of cbl; increasing the concentration of PRL to 100 nM had only a minimal effect upon the phosphorylation of cbl. The cbl protein appears to be constitutively associated with grb2 and the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase). The constitutive association of cbl with the p85 subunit of PI 3-kinase was observed in Nb2 cells as well as in 32Dcl3 cells transfected with either the rat Nb2 (intermediate) form of the PRL receptor or the long form of the human PRL receptor. A glutathione S-transferase fusion protein encoding the SH3 domain of the p85 subunit of PI 3-kinase bound to cbl in lysates of both unstimulated and PRL-stimulated Nb2 cells; however, neither of the SH2 domains of p85 bound to cbl under the same conditions. PRL stimulation increased the cbl-associated PI kinase activity. The majority of PI kinase activity appeared to be cbl-associated after PRL stimulation. These results suggest that cbl may function as an adapter protein in PRL-mediated signaling events and regulate activation of PI 3-kinase. Our model suggests that the p85 subunit of PI 3-kinase is constitutively associated with cbl through binding of the p85 SH3 domain to a proline-rich sequence in cbl. After the tyrosine phosphorylation of cbl, an SH2 domain(s) of p85 binds to a specific phosphorylation site(s) in cbl, leading to the activation of PI 3-kinase.
用催乳素刺激Nb2细胞会导致一种120 kDa的蛋白质在酪氨酸残基上快速磷酸化，该蛋白质被鉴定为衔接蛋白cbl。cbl的最大磷酸化发生在催乳素刺激后20分钟，随后下降。低至5 nM的催乳素刺激即可导致cbl磷酸化；将催乳素浓度增加到100 nM对cbl磷酸化的影响极小。cbl蛋白似乎与grb2以及磷脂酰肌醇3激酶（PI 3激酶）的p85亚基组成性结合。在Nb2细胞以及转染了大鼠Nb2（中间）形式的催乳素受体或人催乳素受体长形式的32Dcl3细胞中都观察到了cbl与PI 3激酶p85亚基的组成性结合。编码PI 3激酶p85亚基SH3结构域的谷胱甘肽S转移酶融合蛋白在未刺激和催乳素刺激的Nb2细胞裂解物中均与cbl结合；然而，在相同条件下，p85的两个SH2结构域均未与cbl结合。催乳素刺激增加了与cbl相关的PI激酶活性。催乳素刺激后，大多数PI激酶活性似乎与cbl相关。这些结果表明，cbl可能在催乳素介导的信号转导事件中作为衔接蛋白发挥作用，并调节PI 3激酶的激活。我们的模型表明，PI 3激酶的p85亚基通过p85 SH3结构域与cbl中富含脯氨酸的序列结合而与cbl组成性结合。cbl酪氨酸磷酸化后，p85的一个或多个SH2结构域与cbl中的特定磷酸化位点结合，从而导致PI 3激酶激活。
