[Date Prev][Date Next][Thread Prev][Thread Next][Date Index][Thread Index]

kinases Protein kinase band shifts



To those of you out there that have used Western blotting to look at the
regulation of protein kinases, I would greatly appreciate your help. We
have been developing a technique in my laboratory to monitor the regulation
of over 50 different protein kinases at once on a single SDS-PAGE gel. The
method exploits the bandshifts that often occurs when protein kinases and
their substrates undergo phosphorylation. So far, in diverse model systems,
we have detected band shifts in the following protein-serine kinases:
cAMP-dependent protein kinase, cGMP-dependent protein kinase,
PKBalpha(Akt1), PKBbeta(Akt2), PKCbeta, PKCzeta, PKCepsilon, Erk1, Erk2,
Rsk1, Rsk2, p70 S6 kinase, p46 JNK(SAPK), p54 JNK(SAPK), Pak1, Mek4, Mek6
and casein kinase 2. We have also seen very small bandshifts in p38alpha
(Hog) MAP kinase. We have failed to see band-shifts in Mek1 and MAPKAPK2
under conditions where these kinases should have been phosphorylated and
activated.

I would be very grateful to learn from any of you whether you are aware of
any other protein-serine kinases or protein-tyrosine kinases that exhibit
bandshifting on SDS-PAGE gels, the model system where the bandshift was
seen and what stimulus was used. For those that send me an e-mail reply
with such information, I will send back a list of the findings that members
of this discussion group have provided. If enough people participate and
the list is large, then I will transmit it to everyone (anonymity would be
maintained).

I believe that such band shift assays for kinase changes are an excellent
way to probe whether kinases are regulated in different model systems.
Kinases that display such changes can be further examined with
phospho-specific antibodies or in phosphotransferase activity assays. If
all goes well, then I may be able to provide such analysis as a service
those in the signal transduction community.

Thank you from Steven Pelech.

Steven Pelech, Ph.D.
Professor
Department of Medicine
Rm S125, 2nd Floor, Koerner Pavilion
2211 Wesbrook Mall
University of British Columbia
Vancouver, B.C.
Canada  V6T 1Z3

Office phone: (604) 822-8086
Office facsimile: (604) 822-8693
Confidential home facsimile: (604) 272-2102
E-mail: spelech@home.com