PROTEIN-TYROSINE PHOSPHORYLATION DURING SEA-URCHIN FERTILIZATION - MICROTUBULE DYNAMICS REQUIRE TYROSINE KINASE-ACTIVITY

Protein tyrosine phosphorylation plays an important role in cell growt h, mitosis, and tumorigenesis. It has also been implicated in meiotic maturation and fertilization. We have used anti-phosphotyrosine immuno fluorescence and immunoblotting to identify sperm and egg proteins whi ch are phosphorylated on tyrosine residues prior to and during sea urc hin fertilization, On immunoblots of sperm proteins, the monoclonal an ti-phosphotyrosine antibody detected three major proteins with molecul ar weights of 44, 82, and 100 kD, and six minor bands at 46, 48, 70, 7 6, 95, and 150 kD. These phosphotyrosyl proteins were localized to the sperm acrosomal and centriolar fossae. In contrast, staining was foun d globally in unfertilized eggs, and the antibody recognized two major egg phosphotyrosyl proteins of molecular weights 42, and 50 kD, and f ive minor bands at 40, 90, 116, 130, and 150 kD. While immunofluoresce nt staining remained throughout the fertilized egg cytoplasm, there we re dynamic changes in the staining intensity of single bands. The 90 k D immunoreactive band increased in intensity, and the 40 and 32 kD ban ds disappeared by 15 min after fertilization. Loss of the 40 and 42 kD bands was due to dephosphorylation by okadaic acid-sensitive phosphat ase(s). The 50 kD immunoreactive protein was unchanged up to the 8-cel l stage and was still present in blastulae, indicating its importance throughout fertilization and early development. Alterations in the pat tern of phosphotyrosine-containing proteins during fertilization did n ot depend on nascent proteins and could not be completely mimicked by increasing intracellular calcium, pH, and protein kinase C activity al one. Since changes in the fertilization pattern of phosphotyrosyl prot eins occurred during formation of the sperm aster and mitotic spindle, we analyzed the role of protein tyrosine kinase activity in these pro cesses using the tyrosine kinase specific inhibitor, erbstatin. Both t he sperm aster and mitotic spindle were disrupted, indicating an invol vement of tyrosine phosphorylation in these processes during interphas e and mitosis. We conclude that the changes in phosphotyrosyl proteins play an important role in fertilization and early development of sea urchin eggs. Control of microtubule assembly into the sperm aster and mitotic spindle of the first cell cycle are examples of such roles. (C ) 1995 Wiley-Liss, Inc.