Lim T, Sossin W S
Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, BT 110, 3801 University Street, Montreal, Quebec, Canada.
Neuroscience. 2006 Aug 11;141(1):277-85. doi: 10.1016/j.neuroscience.2006.03.063. Epub 2006 May 4.
In Aplysia, persistent increases in synaptic strength are paralleled by the persistent activation of the novel protein kinase C Apl II. We raised a phosphospecific antibody against serine 725, the hydrophobic motif in protein kinase C Apl II. Phosphorylation of serine 725 increased in parallel to the persistent activation of the kinase. We expressed protein kinase C where this site was mutated to an alanine to prevent phosphorylation. The mutated protein kinase C showed decreased specific activity consistent with a model where the kinase is less stable in the absence of phosphorylation of this site. Endogenous phosphorylation of protein kinase C Apl II at serine 725 was unaffected by either activation of protein kinase C by phorbol esters, or inhibition of protein kinase C using two distinct inhibitors, suggesting the site is not autophosphorylated. Consistent with this, overexpressed kinase-dead protein kinase C Apl II still was phosphorylated at serine 725, although to a lesser extent than wild-type protein kinase C Apl II. While PDK appears to interact with the serine 725 site, it is not responsible for its phosphorylation. Finally inhibition of phosphoinositide-3 kinase or the target of rapamycin by pharmacological agents did not block basal phosphorylation of serine 725 in Aplysia ganglia. Our results suggest trans-phosphorylation of protein kinase C Apl II as Ser 725 occurs during persistent activation of the kinase, but this does not appear to be downstream of phosphoinositide-3 kinase.
在海兔中，突触强度的持续增加与新型蛋白激酶C Apl II的持续激活同时出现。我们制备了一种针对丝氨酸725的磷酸特异性抗体，丝氨酸725是蛋白激酶C Apl II中的疏水基序。丝氨酸725的磷酸化与激酶的持续激活平行增加。我们表达了该位点突变为丙氨酸以防止磷酸化的蛋白激酶C。突变的蛋白激酶C显示出比野生型更低的比活性，这与该位点未磷酸化时激酶稳定性较低的模型一致。佛波酯激活蛋白激酶C或使用两种不同抑制剂抑制蛋白激酶C均未影响蛋白激酶C Apl II在丝氨酸725处的内源性磷酸化，这表明该位点不是自身磷酸化的。与此一致的是，过表达的激酶失活型蛋白激酶C Apl II在丝氨酸725处仍然被磷酸化，尽管程度低于野生型蛋白激酶C Apl II。虽然PDK似乎与丝氨酸725位点相互作用，但它并不负责该位点的磷酸化。最后，用药物抑制磷酸肌醇-3激酶或雷帕霉素靶蛋白并不阻断海兔神经节中丝氨酸725的基础磷酸化。我们的结果表明，在激酶的持续激活过程中发生了蛋白激酶C Apl II丝氨酸725的反式磷酸化，但这似乎不在磷酸肌醇-3激酶的下游。
