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The molecular basis of Src kinase specificity during vertebrate mesoderm formation
Joanne Hama1, Crystal Suri, Tomomi Haremaki
1Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Abstract:
Members of the Src family of non-receptor tyrosine kinases play a critical role in mesoderm formation in the frog, Xenopus laevis, acting as required mediators downstream of the fibroblast growth factor receptor. At least four members of this gene family, Src, Fyn, Yes, and Laloo, are expressed during early embryonic development. Ectopic expression of Laloo and Fyn, but not Src, induce mesoderm in ectodermal explants, indicating that these factors are non-redundant during early vertebrate development. Here we investigate the basis for the differential activity of the Src and Laloo kinases during mesoderm formation. We demonstrate that although both Src and Laloo physically interact with the substrate protein SNT-1/FRS2alpha only Laloo phosphorylates SNT-1, an event previously shown to be required for the activity of the latter and for mesoderm induction in vivo. We show that Src is enzymatically capable of stimulating mesoderm formation, as an activated Src construct both phosphorylates SNT-1 and induces mesoderm in explant cultures. However, a chimeric Laloo construct containing a Src C-terminal tail is inactive, suggesting that the early embryo contains a specific Laloo-activating, or Src-inactivating, factor. Finally, through further chimeric analysis, we provide evidence to suggest that differences in Laloo and Src activity are also mediated by the SH2, SH3, and kinase domains of these molecules.
Insights
Src and Laloo kinases are crucial for mesoderm formation in Xenopus. While both interact with SNT-1, only Laloo phosphorylates it, driving mesoderm induction, revealing non-redundant roles in development.
Area of Science:
- Developmental Biology
- Molecular Biology
- Cell Signaling
Background:
- Src family kinases are essential for mesoderm formation in Xenopus laevis, downstream of fibroblast growth factor receptor signaling.
- Four Src family members (Src, Fyn, Yes, Laloo) are expressed during early Xenopus development.
- Ectopic expression studies reveal non-redundant roles for Fyn and Laloo, but not Src, in inducing mesoderm.
Purpose of the Study:
- To investigate the differential activity of Src and Laloo kinases in mesoderm formation.
- To elucidate the molecular mechanisms underlying the distinct roles of Src and Laloo in early vertebrate development.
Main Methods:
- Investigated physical interactions between Src, Laloo, and the substrate SNT-1/FRS2alpha.
- Assessed the phosphorylation activity of Src and Laloo on SNT-1.
- Utilized activated Src constructs and chimeric constructs of Laloo and Src to analyze kinase function.
- Employed ectodermal explant cultures to study mesoderm induction in vivo.
Main Results:
- Both Src and Laloo interact with SNT-1/FRS2alpha, but only Laloo phosphorylates it, which is essential for mesoderm induction.
- Activated Src can phosphorylate SNT-1 and induce mesoderm, indicating enzymatic capability.
- A chimeric Laloo construct with a Src C-terminal tail lost activity, suggesting an embryonic factor regulates Src/Laloo activity.
- Chimeric analysis indicated that differences in Src and Laloo activity are also influenced by their SH2, SH3, and kinase domains.
Conclusions:
- Laloo's specific phosphorylation of SNT-1 is critical for mesoderm induction, highlighting its non-redundant role.
- While Src is enzymatically capable, its activity in mesoderm formation is differentially regulated compared to Laloo.
- Specific regulatory factors and domain interactions within the Src family contribute to their distinct functions during early vertebrate development.