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First published online November 24, 2004
doi: 10.1242/10.1242/jcs.01547

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Distinct roles for multiple Src family kinases at fertilization

Forest J. O'Neill^*, Jessica Gillett^* and Kathy R. Foltz

Department of Molecular, Cellular and Developmental Biology and the Marine Science Institute, University of California, Santa Barbara, CA 93106-9610, USA

View larger version (61K): [in a new window]   Fig. 1. Alignment of the deduced amino acid sequence of the AmSFK1, AmSFK2 and AmSFK3 cDNAs with that of human Src (GenBank accession number P12931). Dashes indicate identity and dots represent gaps in the alignment. The unique N-terminal domains are followed by the SH3 domain (green), the SH2 domain (red) and the kinase domain (blue) (Xu et al., 1999). The conserved tyrosines of the activation loop (huSrc Tyr419) and the C-terminus (huSrc Tyr527) are indicated by asterisks. Key residues in the Src SH2 domain that are thought to contribute to binding specificity are indicated in bold font and predicted structural assignments ( helices and ß strands) are indicated (Kuriyan and Cowburn, 1997). The C-terminal sequence of huSrc that serves as the antigen for the sc-18 antibody (see Materials and Methods) is boxed.

View larger version (16K): [in a new window]   Fig. 2. Phylogenetic analysis of echinoderm members of the Src superfamily. The phylogenetic tree was constructed using the neighbor-joining method based on the proportion of amino acid differences (see Materials and Methods). Echinoderm sequences are boxed and shaded.

View larger version (104K): [in a new window]   Fig. 3. Developmental expression of A. miniata SFK transcripts. A blot of 1 µg of polyA+ mRNA from A. miniata eggs (E) and embryos at different stages (B, blastula; G, gastrula) and bipinnaria larvae (Bi) was probed with the radiolabeled 5' UTR of AmSFK1 cDNA, the 3' UTR of the AmSFK2 cDNA or the 3' UTR of the AmSFK3 cDNA (top panels). The bottom panels are photographs of the stained RNA gel prior to transfer, demonstrating that each lane contained the same amount of polyA+ RNA.

View larger version (37K): [in a new window]   Fig. 4. Specificity of AmSFK antibodies. In vitro transcription and translation in the presence of [35S]methionine of each full-length AmSFK cDNA (input) was followed by immunoprecipitation (IP) with anti-vertebrate Src A sc-18 (sc), anti-AmSFK1 (1), anti-AmSFK2 (2) or anti-AmSFK3 (3). IPs were separated on a 12% polyacrylamide gel. The gel was stained, dried and exposed to x-ray film (40 hour exposure shown). Molecular weight markers are indicated in kDa.

View larger version (57K): [in a new window]   Fig. 5. Characterization of AmSFK proteins in eggs. Cytosolic (C) and membrane (M) fractions of A. miniata eggs (20 µg per lane) were separated on a 12% polyacrylamide SDS gel, transferred to nitrocellulose and probed with AmSFK1 IgY, AmSFK2 IgY or AmSFK3 IgY. Antibodies were detected with HRP-conjugated anti-chicken and ECL for a 30 second exposure. Arrows indicate the AmSFK proteins. Positions of molecular weight markers are indicated in kDa. The results are representative of nine experiments.

View larger version (58K): [in a new window]   Fig. 6. Purified GST-SH2 domain fusion proteins used for microinjections. Proteins were purified from induced bacterial lysates, dialyzed and concentrated. 3 µg protein were loaded on a 10% polyacrylamide gel and were stained with Coomassie Blue. Molecular weight markers are indicated in kDa.

View larger version (14K): [in a new window]   Fig. 7. Effects of injection of AmSFK SH2 domain proteins on Ca2+ release at fertilization. A. miniata oocytes were injected with the indicated protein mixed with Calcium Green dextran (CaG), matured and then fertilized. For each SH2 domain, 25 µM is equivalent to 1 mg/ml. Traces show Calcium Green fluorescence (y-axis) as a function of time (x-axis). Asterisks indicate the time of the fertilization potential, which marks sperm-egg fusion (McCulloh and Chambers, 1992; Swann et al., 1992).

View larger version (31K): [in a new window]   Fig. 8. Kinase activity of AmSFK1. (A) Unfertilized (U) and fertilized eggs (F) 1, 2 or 3 minutes post sperm addition, were lysed in a buffer containing Triton X-100 and total soluble protein was subjected to immunoprecipitation with the AmSFK antibodies followed by immune complex assays in the presence of unlabeled and [-32P]ATP. Labeled proteins were separated on 12% polyacrylamide gels, which were stained, dried and exposed to x-ray film. As a mock control (M), egg lysates were precipitated with anti-IgY beads alone. Arrow indicates the Src substrate. The position of the IgG heavy chain from the secondary antibody beads used in the immunoprecipitations, which was weakly and non-specifically labeled in the assays, is indicated. (B) Representative time course (post sperm addition) of AmSFK1 immune complex kinase assay (ICKA) as assayed by labeling of the Src substrate. (C) Anti-AmSFK1 immunoblot of the inputs used in the immune complex kinase assay shown in panel B. (D) Graph representing the average relative increase in AmSFK1 activity over time as assessed by substrate labeling normalized to the zero time point (n=4). Error bars represent standard deviation; increased activity at the 60 second and later time points are statistically significant compared to that at the zero time point (P 0.001).

View larger version (32K): [in a new window]   Fig. 9. Activity of AmSFK1 and AmSFK3 during the first 2 minutes of fertilization. (A) Unfertilized (time 0) and fertilized eggs (time indicated is post-sperm addition) were lysed in a buffer containing Triton X-100 and total soluble protein was subjected to immunoprecipitation with AmSFK antibodies followed by immune complex assays in the presence of [-32P]ATP and the absence of cold carrier, to increase sensitivity. Labeled proteins were separated on 12% polyacrylamide gels, which were stained, dried and exposed to x-ray film. Representative autoradiographs after 9 hours' exposure show peptide substrate labeling over time. (B) Representative autoradiographs of radiolabeled, presumably autophosphorylated, AmSFK1 and AmSFK3 in unfertilized eggs (time 0) and at the point of peak activity in assays lacking exogenously added peptide substrate. (C) Graph representing the average relative increase in AmSFK1 (filled circles) and AmSFK3 (open circles) activity over time as assessed by Src peptide substrate labeling normalized to the zero time point (n=3). Error bars represent standard deviation; for AmSFK1, relative labelling at the 45 second and later time points is statistically significant compared to that at the zero time point; for AmSFK3, labelling at the 10, 20, 30 and 45 second time points is significant compared to that at the zero time point (P 0.001).

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