Amphibian oocytes are large cells, 1.3 mm in diameter that are suitable for microinjection. Both insulin and progesterone induce phosphorylation of ribosomal protein S6 as well as meiotic maturation. Microinjection of partially purified insulin receptor tyrosine kinase or purified pp60v-src, the tyrosine kinase of Rous sarcoma virus transformed cells, leads to S6 phoshorylation on serine residues. It is proposed to identify intermediates between tyrosine kinase activity of injected proteins and increased S6 phosphorylation. S6 protein kinases and phosphatases will be purified from oocytes and eggs and tested for ability to serve as substrate for tyrosine protein kinases as well as for ability to affect S6 phosphorylation and protein synthesis after injection into living oocytes. Other studies will examine 2-dimensional gels of phosphorylated protein from tyrosine kinase injected oocytes to identify phosphotyrosyl containing proteins, and such proteins will be purified on affinity columns containing a monoclonal antibody against phosphotyrosine that is known to specifically absorb phosphotyrosine containing proteins. Antisera that recognize the xenopus oocyte normal cellular homolog of pp60v-src will be used to examine possible changes in oocyte cell src during maturation. The activities of affinity purified insulin receptor kinase or IGF, receptor kinase will be examined after injection into oocytes to determine the extent of metabolic changes with injected receptor kinase. These studies will provide new insights into the role of tyrosine phosphorylation in the mechanism of action of transforming gene products as well as hormones that act via receptors that are tyrosine kinases.
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