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kinases protein kinase band shifts



Many thanks to those of you responded to my request for the names of
protein kinases that show evidence of band shifts (due to phosphorylation)
on SDS-PAGE gels. So far the list of protein kinases that can display
altered mobilities on SDS-PAGE gels includes:

casein kinase-2
Cdc2 (Cdk1)
Cdk2
cAMP-dependent protein kiinase
cGMP-dependent protein kinase
dual leucine zipper kinase DLK
Eg2
Erk1 and Erk2
hCk1 and hCK2 (human checkpoint kinases)
IKK-alpha and IKK-beta
Mek1
Mek4
Mek6
Myt1
p38 Hog MAP kinase
p70 S6 kinase (also p54 S6 kinase variant)
Pak1
PDK1
PKBalpha (Akt1)
PKBbeta (Akt2)
protein kinase C (all isoforms)
Raf1
Rsk1, 2 and 3 isoforms
SAPK (JNK) p46 and p54 isoforms
Wee1

If any of you are aware of any other kinases that band shift, please let me
know. I am sure that many more protein kinases undergo mobility shifts when
they are phosphorylated.

Personally, I feel that quantitation of the amount of immunoreactive
phosphorylated and dephosphorylated forms of a kinase are a better measure
of the degree of activation of that kinase than can be obtained with
phospho-specific antibodies. It also has the advantage that pre-labelling
of cells with radioactive phosphate is unnecessary to study the
phosphorylation state of proteins. The basic problem with
phospho-site-specific antibodies, apart from cost, is that it is not
possible to obtain quantitative data about how much of a target kinase is
actually phosphorylated (not unless one has a positive control where a
stimulus is also used that can effect a 100% activation of that kinase). It
is also debatable how specific many phospho-site antibodies may actually
be. With about another 1400 or so kinases yet to be identified, it is hard
to rule out that these phosphorylation sites are not in other protein
kinases or even other kinase substrates. I recommend that investigators
look for band shifts in the kinases that they are interested in first,
before they turn to phospho-specific antibodies or phosphotransferase
activity assays.

It is intriguing that a few protein kinases (e.g. PKB, p70 S6 kinase, Rsk
isoforms) display multiple bands on Western blots. These bands appear to
correspond to multiple phosphorylation states. However, it may take several
phosphorylation events to induce a band shift to a new level. In a way, it
is as if proteins have quantum states of phosphorylation that can be
discerned on an SDS-PAGE gel. It is possible that each quantum state
represents a unique set of phosphorylations perhaps catalyzed by one or
more protein kinases. Therefore, by monitoring the band shift level of a
protein kinase or it substrate, it may be possible to evaluate whether it
has been phosphorylated by a particular set of kinases. This is especially
evident for p70 S6 kinase. Following rapamycin-treatment of cells, p70 S6
kinase is essentially only found in its highest mobility, lower most band
(presumably the dephosphorylated state of p70 S6 kinase). Conversely, in
mitogen-treated cells, three other bands of lower mobility for p70 S6
kinase are observed, with most of the kinase found in the highest band
form. Interestingly, in response to the Mek1 inhibitor PD98059, the p70 S6
kinase is predominantly found in the intermediate phosphorylated forms. I
interpret these findings to indicate that a FRAP/mTOR-mediate
phosphorylation of p70 S6 kinase is required before it can be
phosphorylated by any other kinases. Following this initial phosphorylation
event, it may become phosphorylated by other protein kinases, including
those that are downstream from Mek1, which are presumably ERK1 and ERK2,
and by other kinases such as PDK1.

I would be curious to learn the thoughts of others on this matter.

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