Src kinase assay - (Jan/15/2008 )

Hi!

I have to carry out a Src kinase assay using enolase as substrate. Do you have some protocol to assay this enzime?? Do you think it would be better to use another protocol?

Thank you!!

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it depends on what you like to analyze: Do you want to determine Src phosphotransferase activity or the Src-dependent phosphorylation sites of enolase?

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Hi!

I observe that when I treat cells, there is an increase in both Src Tyr416 and Tyr527 phosphorylation...
Because of Y416 is suposed to be an activator residue and Y527 is an inhibitor residue, I wonder what is happening... so I want to analyze Src kinase activity in order to know the real state of Src (is Src activated or inhibited?)

Thank you for your help and your answers!!

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we often observe the phenomenon you describe; a high phospho-Y416 does not necessarily mean a low phospho-Y527 and vice versa; you may also observe neither a phosphorylation of Y416 nor Y527;

phosphorylation at Y527 means that it is not phosphorylatable at Y418; if Y527 is not phosphorylated it is ready to be (auto)phosphorylated at Y418 but must not necessarily be phosphorylated at Y416;

one has to consider the activities of Csk, PTP1B (for Y527), and interaction of SH2/SH3 domains with CAS or FAK as well as activities of other PY-phosphatases such as PTPalpha, SHP1 and SHP2;

another thing is if you analyze total lysate and not subcellular fractions you get a crude mixture of various Src-containing signalosomes (= various subcellular-specific states of activation)

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