B cell activation and differentiation are dependent on ligand binding to the antigen receptor, membrane immunoglobulin (mIg), which transduces a signal across the plasma membrane that activates both the phosphoinositide pathway and a mIg-regulated protein tyrosine kinase (PTK). Activation of these biochemical pathways is required for subsequent gene transcription and entry into the cell cycle. In addition, we recently demonstrated that expression of a phosphotyrosine phosphatase (PTPase), CD45, is required for signaling following mIg ligation. Thus, based on the above observations it is apparent that mIg-mediated signal transduction is regulated by the dynamic interplay between a PTK and a PTPase (CD45). We propose to examine the physiologic role of CD45 in regulation of B cell activation following antigen receptor ligation. Studies will be carried out to gain a better understanding of the physical interaction that occurs between CD45 and the antigen receptor complex. Co-capping and co- modulation experiments as well as chemical crosslinking studies will be performed to document functional associations between these proteins in the plasma membrane. Further, we will determine whether CD45 expression is necessary for PTK activation. Identification of the specific PTK associated with the antigen receptor complex by coimmunoprecipitation with mIg or its associated proteins (MB-1 and B29) will enable us to determine how its activity is regulated by tyrosine phosphorylation, and what role CD45 plays in this process. Additional experiments will be carried out to determine whether CD45 regulates the tyrosine phosphorylation of specific residues on MB-1 and B29 following their isolation from CD45-variant cell lines. Differences in tyrosine phosphorylation will be correlated with altered function by examining the ability of these proteins to couple mIg to the PTK in CD45-variant cell lines. Finally, we will utilize site-directed mutagenesis of the catalytic domain of CD45 in order to gain a better understanding of the structure-function relationships which control the activity of this PTPase. Mutagenized forms of CD45 will be examined for their ability to reconstitute signal transduction in CD45-negative cells. These studies will contribute significantly to our understanding of the molecular basis of B cell activation and will provide the foundation for further studies concerning the regulation of cell function by tyrosine phosphorylation/dephosphorylation. Specifically, it has been suggested that PTPase expression is critical for the prevention of cellular transformation by counterbalancing the effects of PTK's on cellular processes. Thus, the studies proposed above may provide information that enhances our understanding of the events which result in aberrant activation of the B cell.
| Justement, L B (2000) Signal transduction via the B-cell antigen receptor: the role of protein tyrosine kinases and protein tyrosine phosphatases. Curr Top Microbiol Immunol 245:Jan-51 |
