Singh S S, Chauhan A, Brockerhoff H, Chauhan V P
NYS Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314.
Cell Signal. 1994 Mar;6(3):345-53. doi: 10.1016/0898-6568(94)90039-6.
Phosphatidylinositol 4,5-bisphosphate (PIP2) activates protein kinase C (PKC) in the presence of phosphatidylserine and calcium. Recently it has been demonstrated that direct interaction of PKC with PIP2 in the absence of divalent cation inactivates this kinase. In the present study, the interaction of natural aliphatic polyamines with phosphoinositides was investigated for its possible relevance to PKC-mediated protein phosphorylation. PKC/phosphoinositide interaction was studied by monitoring the changes in (a) intrinsic fluorescence of the enzyme, and (b) PKC activity (protamine sulphate or histone III-S as substrate). All the phosphoinositides: PIP2, phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol (PI) inactivated PKC with an IC50 of 0.4 microM for PIP2, 5 microM for PIP and 10 microM for PI. Hydrogenated PIP2 behaved similarly to that of natural PIP2. Time-dependent studies showed very rapid inactivation of PKC by PIP2. The polyamines spermine and spermidine at physiological concentrations protected PKC from phosphoinositides-mediated inactivation when added prior to PKC interaction with phosphoinositides. Putrescine was least effective. Addition of spermine or spermidine to PKC/phosphoinositides incubation mixture did not reverse PKC activity indicating that the inactivation of PKC by phosphoinositides is irreversible. Fluorescence quenching experiments showed that phosphoinositides inactivate PKC by inducing conformational changes of the enzyme that are prevented by spermine. We propose that polyamines protect PKC and possibly other protein kinase from phosphoinositides-mediated inactivation, and that inactivation of protein kinases by phosphoinositides may not have physiological relevance.
磷脂酰肌醇4,5-二磷酸（PIP2）在磷脂酰丝氨酸和钙存在的情况下激活蛋白激酶C（PKC）。最近有研究表明，在没有二价阳离子的情况下，PKC与PIP2的直接相互作用会使该激酶失活。在本研究中，研究了天然脂肪族多胺与磷酸肌醇的相互作用，探讨其与PKC介导的蛋白质磷酸化的可能相关性。通过监测以下变化来研究PKC/磷酸肌醇相互作用：（a）酶的内在荧光，以及（b）PKC活性（以硫酸鱼精蛋白或组蛋白III-S为底物）。所有的磷酸肌醇：PIP2、磷脂酰肌醇4-磷酸（PIP）和磷脂酰肌醇（PI）都会使PKC失活，PIP2的IC50为0.4微摩尔，PIP为5微摩尔，PI为10微摩尔。氢化PIP2的行为与天然PIP2相似。时间依赖性研究表明，PIP2能非常迅速地使PKC失活。当在PKC与磷酸肌醇相互作用之前加入生理浓度的多胺精胺和亚精胺时，它们能保护PKC免受磷酸肌醇介导的失活。腐胺的效果最差。向PKC/磷酸肌醇孵育混合物中添加精胺或亚精胺并不能恢复PKC活性，这表明磷酸肌醇对PKC的失活是不可逆的。荧光猝灭实验表明，磷酸肌醇通过诱导酶的构象变化使PKC失活，而精胺可阻止这种变化。我们提出，多胺可保护PKC以及可能的其他蛋白激酶免受磷酸肌醇介导的失活，并且磷酸肌醇对蛋白激酶的失活可能没有生理相关性。
