Anti‐peptide Antibody Identifies a 57 kDa Protein Tyrosine Kinase in the Sea Urchin Egg Cortex

Abstract

The egg plasma membrane and cortical structures are highly enriched in protein tyrosine kinase activity which is thought to play an important role in the fertilization process. In order to identify the tyrosine protein kinases in the egg cortex, a site directed polyclonal antibody was produced against a peptide duplicating a conserved region of the catalytic domain of the sea urchin c-abl gene product. The region chosen as an antigen had a high degree of homology (57%) to other protein tyrosine kinases. The antibody was found to bind with a high degree of specificity to a 57 kDa protein tyrosine kinase in S. purpuratus eggs. The antibody was capable of immunoprecipitating the enzyme as a 57 kDa phosphoprotein from purified egg cortex fractions solubilized in NP-40. lmmunoprecipitation was completely inhibited by prior incubation of the antibody with the synthetic peptide used as an antigen. Binding of the antibody completely inhibited kinase activity. However, the immunoprecipitated kinase activity could be eluted from the Sepharosecoupled antibody and was shown to have catalytic activity towards a tyrosine containing peptide substrate. The enzyme also underwent autophosphorylation on tyrosine in vltro. Ultrastructural localization of the kinase by immuno-electron microscopy revealed that the enzyme was primarily restricted to the egg plasma membrane.

Purification and Characterization of a Src-related p57 Protein-tyrosine Kinase from Xenopus Oocytes

“…The enzyme was identified by an antibody against the synthetic peptide corresponding to residues Thr-Tyr-Thr-Ala-Gln-Ala-Gly-AlaLys-Asn-Pro-Ile-Lys-Trp (a part of the IDA region) of the sea urchin c-Abl. They also found that the binding of the antibody completely inhibited the kinase activity (36). These conflicting results concerning the effect of antibody on the kinase activity might be explained by the difference in the epitopes that are recognized by these anti-IDA antibodies.…”

“…As to the effect of the antibody on the kinase activity, Casnellie et al (35) reported that the Lck activity was inhibited by their antibody. Recently, Peaucellier et al (36) identified a 57-kDa protein-tyrosine kinase in sea urchin eggs. The enzyme was identified by an antibody against the synthetic peptide corresponding to residues Thr-Tyr-Thr-Ala-Gln-Ala-Gly-AlaLys-Asn-Pro-Ile-Lys-Trp (a part of the IDA region) of the sea urchin c-Abl.…”

“…3 The major epitope for the anti-pepY antibody is shown to reside in the the carboxyl-terminal half of the IDA region of Src (13). The antibody used by Casnellie et al (36) was raised against a peptide that lacks seven carboxyl-terminal amino acids when aligned with our pepY. Thus, the resulting antibody would not be able to bind to the carboxyl-terminal half of the IDA region of Lck.…”

“…Their observations indicate the importance of tyrosine phosphorylation for the ongoing of fertilization. As a candidate for the protein-tyrosine kinase being responsible for the increased tyrosine phosphorylation of proteins at fertilization, a 57-kDa tyrosine kinase (36) and an Abl-related 220-kDa tyrosine kinase (48) have been reported. Both kinases are localized to the egg cortex (36,48).…”

“…As a candidate for the protein-tyrosine kinase being responsible for the increased tyrosine phosphorylation of proteins at fertilization, a 57-kDa tyrosine kinase (36) and an Abl-related 220-kDa tyrosine kinase (48) have been reported. Both kinases are localized to the egg cortex (36,48). Recently, Kinsey reported a transient kinase activation and time-dependent phosphorylation and dephosphorylation of the 57-kDa kinase on both tyrosine and threonine residues after fertilization (49).…”