autophosphorylation question

Thu Aug 3 19:44:45 EST 1995

As part of my masters thesis research, I am studying a cytoplasmic
tyrosine kinase.  Studies on this kinase have shown a positive correlation
between its level of tyrosine phosphorylation and its kinase activity.
Most of these studies involve immunoprecipitating the kinase and measuring
the immune complex kinase activity by the incorporation of 32-P onto the
exogenous substrate poly (Glu,Tyr).  But some studies use autophosphorylation
as an indication of kinase activity.

My question is:  Is autophosphorylation an accurate measure of tyrosine
kinase activity?

It seems to me it would not be.  Say that the kinase immunopreciptated
from cells treated with substance A is significantly less tyrosine
phosphorylated than kinase from control cells.  If the level of tyrosine
phosphorylation is indeed correlated with kinase activity, one would expect
the kinase immunoprecipitated from control cells to be more active.

In an autophosphorylation assay, wouldn't the less phosphorylated kinase
sample have more "free" tyrosines for the addition of 32-P?  Since no
PTPases should be present in the immunoprecipitates, shouldn't there be
little phosphate turnover?  So wouldn't the more phosphorylated kinase 
have  less incorporation of 32-P?

My current thinking is that autophosphorylation tells nothing about the
actual kinase activity.

Am I looking at this the right way?  I would appreciate any input.

Thank you.
Peter J. Bartholomew


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