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Re: PKC BetaII



The dominant negative mutant protein is probably reducing the
phosphorylation of the endogenous protein that is required for activation,
by competing with the endogenous protein for the activating kinase.

The antisense message might be enhancing activation of the PKC by a stress
mechanism.  Alternatively, your assay may be picking up another kinase.
How are you assaying for the PKC beta II activity (how specific is your
assay), and are you immunoprecipitating?  If your assay is fairly specific,
a third alternative is that there is a significant enhancement of kinase
activity in cells that have antisense, presumably via a double stranded
mRNA activated kinase.  If this activates PKCbetaII, then your may see a
lot of activity, even though the protein is reduced (I assume the PKCbetaII
detectable by Western in your antisense transfected cells is decreased).

Katheryn Resing


>Dear researchers
>
>I have got H441 (lung adenocarcinoma) cells stably transfected with
>plasmid constructs having(1) antisense cDNA of dominant negative mutant of
>PKC beta II and (2) dominant neagtive mutant mutant of PKC beta II. As
>expected, cells transfected with Dominant negative PKC beta II
>construct(2) showed significantly reduced PKC beta II activity. On the
>other side cells transfected with construct having antisense cDNA of this
>dominant negative mutant (1), showed two fold increase in PKC beta II
>activity. Can anybody help me to understand, what exactly is happening,
>why I am getting increase in PKC beta II activity with antisense construct
>of dominant negative mutant of PKC beta II ?
>
>Thanks
>
>Shanjana Awasthi
>Physiology
>University of Texas Health Sci Ctr at San Antonio
>San Antonio Texas
>e-mail: shanjana@eudoramail.com
>fax: (210)567 4410
>
>
>
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