Signal transduction through the B cell immunoglobulin receptor is now known to involve interconnecting enzyme pathways, catalyzed by tyrosine kinase and phospholipase C. The first enzyme system utilizes 3 src family kinases, Blk, Fyn and Lyn, to phosphorylate a number of proteins on tyrosine residues following receptor crosslinking. One of the substrates has been identified as the proto-oncogene vav. Vav co-precipitates with all of the src kinases found in B lymphocytes and has an intrinsic tyrosine kinase activity associated with it. Another substrate that has been examined is a 72 Kd protein that is phosphorylated when the cells are stimulated with anti-IgD but not anti-IgM, this protein is maximally phosphorylated by approx. 20 seconds and then dephosphorylated by 1 minute. This substrate has not been identified yet but, since, only 1% of phosphorylated proteins are phosphorylated on tyrosine and since this is rapidly dephosphorylated taken together this implies that this 72 Kd protein is potentially a regulatory protein. We are setting up a 2D gel analysis system that will allow us to separate proteins on the basis of molecular weight and isoelectric point. In addition, since the conditions for stimulating the cells are very strict with regard to cell number we have been developing ways to increase the load of protein in the first dimension by doing multiple loads of extract separated by several hours to allow the proteins to partially focus and leave the loading cup before the next addition of sample. In this way we hope to be able to load onto the gel between 20-40 times the amount of cell extract that we routinely load on a 1D gel and this will give us enough protein to get a partial amino acid sequence on for further development.
