Protein phosphorylation occurs in T lymphocytes which are activated to proliferate after MHC-restricted antigen recognition or interaction of IL-2 with its receptor. The various reactions which take place and ultimately instruct the cell to enter S phase are essentially unknown. Under certain conditions, T cell activation causes heightened proteins tyrosine kinase activity, and it is probable that tyrosine kinases are involved in the initiation, and, perhaps, in the propagation of the signal to proliferate. Loss of control of signal transmission may be a factor in development of neoplasms of lymphocytes or other diseases related to hypoplasia or hyperplasia of lymphoid tissue. Our long-term goal is to sort out the reactions which culminate in initiation of DNA synthesis in lymphocytes. In preliminary work we found that particulate fractions of murine EL-4 T cells contained 60kd and 64kd proteins heavily phosphorylated on tyrosine residues. For comparative purposes, we will examine human CEM T cells for similar proteins. It is likely that the 60kd protein is a tyrosine kinase; the 64kd protein may be a tyrosine kinase or the substrate for one. The proposed work also deals with isolation, characterization and comparison of the 60kd phosphorylated proteins derived from both cell lines. We propose to isolate the proteins using combinations of affinity and ion-exchange chromatography, chromatofocusing, and gel filtration, and to compare them with respect to molecular weights, activities as tyrosine kinase, and similarity of their proteolytic cleavage maps. We also intend to isolate the 64kd proteins using appropriate methods selected from those mentioned above, and to compare them according to their potential tyrosine kinase activities, molecular weight and peptide maps. Finally, we plan to identify the major substrate of the isolated 32P-60 kd proteins in the particulate fractions using autoradiography, and to determine if the isolated 64kd proteins are, themselves, substrates which can accept the 32P directly from the 32P-60 kd proteins or from 32P- ATP catalyzed by the 60kd proteins.
