Hello everybody,
My laboratory is working on discovery of new phosphorylation sites in certain partners of a mitotic kinase. To study the relevance of this phosphorylations in the polypeptide chain, we will truncate these positions and study in vitro the potential consequences. Does somebody know at least two possible experiments I could perform to study the functional outcomes of the mutation?
Thank you very much in advance!
Dear Ruben,
Truncations will not tell you that much. Better mutate your target amino acid to D or E and to A. If you are studying a mitotic kinase you should have already some assay. Otherwise you may analyze whether the interaction is affected, e. g. in a pull-down/immunoprecipitation assay.
Best, Uwe
uwe is right, a precise mutagenesis of the phospho site is better than a truncation...ideally nowadays, i would insert mutated (S to A or D or E mutant) gene in a landing pad such aavs1 (dalvai ..doyon lab, cell reports paper) in a crisp KO cell line (if the KO is not lethal) . this way you will probe the functional significance of the phospho site...in a null background..., you can also perfom this by KO transiently you FP with si RNA in the 3UTR and rescue your phenotype, assay .....in other word many things can be done....
Generation of a rescue system with point mutants followed by comparative study with WT rescued cells for the specific kinase-dependent functions (eg. Cell cycle, chromosome alignment etc. as u indicated a mitotic kinase).
Manoj Balakrishna Menon Didier Fesquet Uwe Borgmeyer Thank you very much for your time! Certainly, I did not want to mean "truncation"... my mistake, I wanted to mean just selected mutation of the phosphorylation sites. Your responses are very helpful. Now, as I want to study the effects of these single site mutations, the use of transient silencing of mRNA (although attractive) would not be informative for the phosphorylation blockage, as I would be downregulating the whole product. The comparative study between WT and mutated protein cells is an option. I thought about some assays to observe the functional effects:
•Localization of protein during mitosis
•Midbody organization (my protein is related to late mitosis)
•Microtubule and actin cytoskeleton dynamics
•Intercellular bridge stability
•Generation of multinucleated cells
•Chromosomal aberration
But this is only focused on the mytotic effects. Any other assay that you would add or previously have used?
Thank you again for your time.
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