Wooten M W, Vandenplas M L, Seibenhener M L, Geetha T, Diaz-Meco M T
Department of Biological Sciences, Auburn University, 331 Funchess Hall, Auburn, AL 36849, USA.
Mol Cell Biol. 2001 Dec;21(24):8414-27. doi: 10.1128/MCB.21.24.8414-8427.2001.
Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC-iota becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC-iota were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC-iota in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC-iota were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-iota. Recruitment of PKC-iota into the complex was dependent on the tyrosine phosphorylation state of PKC-iota. The association of src and PKC-iota was constitutive but was enhanced by NGF treatment, with the src homology 3 domain interacting with a PXXP sequence within the regulatory domain of PKC-iota (amino acids 98 to 114). Altogether, these findings support a role for src in regulation of PKC-iota. Tyrosine 256, 271, and 325 were identified as major sites phosphorylated by src in the catalytic domain. Y256F and Y271F mutations did not alter src-induced activation of PKC-iota, whereas the Y325F mutation significantly reduced src-induced activation of PKC-iota. The functional relevance of these mutations was tested by determining the ability of each mutant to support TRAF6 activation of NF-kappaB, with significant impairment by the Y325F PKC-iota mutant. Moreover, when the Y352F mutant was expressed in PC12 cells, NGF's ability to promote survival in serum-free media was reduced. In summary, we have identified a novel mechanism for NGF-induced activation of atypical PKC involving tyrosine phosphorylation by c-Src.
非典型蛋白激酶C（PKC）亚型是神经生长因子（NGF）启动的PC12细胞分化所必需的。在本研究中，我们报告PKC-ι在膜上发生酪氨酸磷酸化，这与用神经生长因子处理后的激活同时发生。PP2和K252a（src和TrkA激酶抑制剂）均以剂量依赖的方式抑制PKC-ι的酪氨酸磷酸化和激活。观察到纯化的src在体外使PKC-ι磷酸化并激活。在src激酶活性缺陷的PC12细胞中，NGF诱导的PKC-ι酪氨酸磷酸化和激活也减弱。此外，我们证明了NGF对src的激活以及包括TrkA受体、src和PKC-ι的信号复合物的形成。PKC-ι募集到复合物中取决于PKC-ι的酪氨酸磷酸化状态。src与PKC-ι的结合是组成性的，但通过NGF处理增强，src同源3结构域与PKC-ι调节结构域内的PXXP序列（氨基酸98至114）相互作用。总之，这些发现支持src在调节PKC-ι中的作用。酪氨酸256、271和325被确定为催化结构域中被src磷酸化的主要位点。Y256F和Y271F突变不改变src诱导的PKC-ι激活，而Y325F突变显著降低src诱导的PKC-ι激活。通过确定每个突变体支持TRAF6激活NF-κB的能力来测试这些突变的功能相关性，Y325F PKC-ι突变体有显著损害。此外，当Y352F突变体在PC12细胞中表达时，NGF在无血清培养基中促进存活的能力降低。总之，我们确定了一种NGF诱导非典型PKC激活的新机制，涉及c-Src介导的酪氨酸磷酸化。
