Liu Y, Liu Y C, Meller N, Giampa L, Elly C, Doyle M, Altman A
Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA.
J Immunol. 1999 Jun 15;162(12):7095-101.
One of the major proteins that is rapidly tyrosine phosphorylated upon stimulation of the TCR/CD3 complex is the 120-kDa product of the c-cbl protooncogene (Cbl). Upon activation, tyrosine-phosphorylated Cbl interacts with the Src homology 2 (SH2) domains of several signaling proteins, e.g., phosphatidylinositol 3-kinase (PI3-K) and CrkL. In the present study, we report that pretreatment of Jurkat T cells with PMA reduced the anti-CD3-induced tyrosine phosphorylation of Cbl and, consequently, its activation-dependent association with PI3-K and CrkL. A specific protein kinase C (PKC) inhibitor (GF-109203X) reversed the effect of PMA on tyrosine phosphorylation of Cbl and restored the activation-dependent association of Cbl with PI3-K and CrkL. We also provide evidence that PKCalpha and PKCtheta can physically associate with Cbl and are able to phosphorylate it in vitro and in vivo. Furthermore, a serine-rich motif at the C terminus of Cbl, which is critical for PMA-induced 14-3-3 binding, is also phosphorylated by PKCalpha and PKCtheta in vitro. These results suggest that, by regulating tyrosine and serine phosphorylation of Cbl, PKC is able to control the association of Cbl with signaling intermediates, such as SH2 domain-containing proteins and 14-3-3 proteins, which may consequently result in the modulation of its function.
TCR/CD3复合物受刺激后迅速发生酪氨酸磷酸化的主要蛋白质之一是原癌基因c-cbl的120-kDa产物（Cbl）。激活后，酪氨酸磷酸化的Cbl与几种信号蛋白的Src同源2（SH2）结构域相互作用，例如磷脂酰肌醇3激酶（PI3-K）和CrkL。在本研究中，我们报道用佛波酯（PMA）预处理Jurkat T细胞可降低抗CD3诱导的Cbl酪氨酸磷酸化，从而降低其与PI3-K和CrkL的激活依赖性结合。一种特异性蛋白激酶C（PKC）抑制剂（GF-109203X）可逆转PMA对Cbl酪氨酸磷酸化的作用，并恢复Cbl与PI3-K和CrkL的激活依赖性结合。我们还提供证据表明PKCα和PKCθ可与Cbl发生物理结合，并能够在体外和体内使其磷酸化。此外，Cbl C末端富含丝氨酸的基序对于PMA诱导的14-3-3结合至关重要，在体外也可被PKCα和PKCθ磷酸化。这些结果表明，通过调节Cbl的酪氨酸和丝氨酸磷酸化，PKC能够控制Cbl与信号中间体的结合，如含SH2结构域的蛋白质和14-3-3蛋白质，这可能会导致其功能的调节。
