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Phosphorylation vs. activity for MAPKs



Dear kinasers,

For ERKs 1 and 2, a single phosphorylation is enough to shift the protein
to its high apparent mw form.  There is a lag between when the first
phosphate goes on and when the second phosphate goes (the phos mechanism is
non-processive, both phosphorylations are partially rate determining, and
both dephosphorylations are partially rate determining....).  So in
principle the correlation between phosphorylation as judged by band shift
and activity as judged by immune complex kinase assay or phospho MAPK
blotting should be imperfect.  In practice all three measures correlate
pretty darn well.

Likewise, the phospho-specific Abs are not perfect measures of MAPK
activity.  The first time we checked the specificities of the Promega
dual-phos specific ERK Ab with ERK2 phosphorylation site mutants, we found
it recognized bis-phos ERK2 and mono-phos-Thr-ERK2 equally well and
essentially did not pick up mono-phos-Tyr-ERK2 or non-phos-ERK2 at all.
>From conversations with colleagues it appears that other lots have been
different in their specificity.

When in doubt do the controls!

Cheers,

Jim
-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-
James E. Ferrell, Jr., M.D., Ph.D.
Associate Professor
Department of Molecular Pharmacology
Stanford University School of Medicine
Stanford CA  94305-5332
ferrell@cmgm.stanford.edu
Phone:  650 725-0765
Fax:  650 725-2952