Puente Lawrence G, Mireau Laura R, Lysechko Tara L, Ostergaard Hanne L
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alta., Canada T6G2S2.
Mol Immunol. 2005 Jun;42(10):1177-84. doi: 10.1016/j.molimm.2004.11.012. Epub 2005 Jan 6.
Protein kinase C (PKC) theta plays a crucial role in T cell activation. We, therefore, examined the regulation of PKCtheta activity in cytotoxic T lymphocytes (CTL). We demonstrated that PMA did not stimulate PKCtheta activation and phospholipase C inhibition did not block anti-CD3-stimulated PKCtheta activation in a CTL clone. This suggests that diacylglycerol is neither sufficient nor required for PKCtheta activation. Furthermore, PKCtheta was only activated in a CTL clone stimulated with plate-bound anti-CD3 but not soluble anti-CD3. However, PMA or cross-linked anti-CD3 stimulated phosphorylation of PKCtheta as measured by a migratory shift, suggesting that phosphorylation was not sufficient for activity. Phosphatidylinositol 3-kinase activity was required for anti-CD3, but not PMA, stimulated phosphorylation and for immobilized anti-CD3-triggered PKCtheta activity. A substantial fraction of PKCtheta was constitutively membrane associated and PMA or CD3 stimulation did not significantly increase membrane association. Our data indicate that phosphorylation of PKCtheta is not a suitable surrogate measurement for PKCtheta activity and that additional, yet to be defined steps, are required for the regulation of PKCtheta enzymatic activity in CTL.
蛋白激酶C（PKC）θ在T细胞活化过程中起着关键作用。因此，我们研究了细胞毒性T淋巴细胞（CTL）中PKCθ活性的调节机制。我们发现，在一个CTL克隆中，佛波酯（PMA）不会刺激PKCθ的活化，而磷脂酶C的抑制也不会阻断抗CD3刺激的PKCθ活化。这表明二酰基甘油对于PKCθ的活化既非充分条件也非必要条件。此外，PKCθ仅在与平板结合的抗CD3刺激的CTL克隆中被激活，而在可溶性抗CD3刺激下则未被激活。然而，通过迁移率变化测定发现，PMA或交联的抗CD3可刺激PKCθ的磷酸化，这表明磷酸化并不足以激活PKCθ。抗CD3刺激的磷酸化以及固定化抗CD3触发的PKCθ活性需要磷脂酰肌醇3激酶活性，但PMA刺激则不需要。相当一部分PKCθ组成性地与细胞膜结合，PMA或CD3刺激并不会显著增加其与细胞膜的结合。我们的数据表明，PKCθ的磷酸化并非PKCθ活性的合适替代指标，在CTL中，PKCθ酶活性的调节还需要其他尚未明确的步骤。
