Interaction of protein kinase C and phosphoinositides: regulation by polyamines.
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.
Singh
S S
SS
NYS Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314.
Chauhan
A
A
Brockerhoff
H
H
Chauhan
V P
VP
D000818:Animals; D001921:Brain / Q000201:enzymology; D004789:Enzyme Activation; D008751:Methylene Blue; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011073:Polyamines / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D011073:Polyamines; D011493:Protein Kinase C; D008751:Methylene Blue