Differential Sensitivity of Src-Family Kinases to Activation by SH3 Domain Displacement

PLOS ONE, Dec 2019

Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo.

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Differential Sensitivity of Src-Family Kinases to Activation by SH3 Domain Displacement

et al. (2014) Differential Sensitivity of Src-Family Kinases to Activation by SH3 Domain Displacement. PLoS ONE 9(8): e105629. doi:10.1371/journal.pone.0105629 Differential Sensitivity of Src-Family Kinases to Activation by SH3 Domain Displacement Jamie A. Moroco 0 Jodi K. Craigo 0 Roxana E. Iacob 0 Thomas E. Wales 0 John R. Engen 0 Thomas E. Smithgall 0 Heinrich Sticht, Universitat Erlangen-Nu rnberg, Germany 0 1 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine , Pittsburgh , Pennsylvania, United States of America, 2 Center for Vaccine Research, University of Pittsburgh School of Medicine , Pittsburgh , Pennsylvania, United States of America, 3 Department of Chemistry and Chemical Biology, Northeastern University , Boston, Massachusetts , United States of America Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by National Institutes of Health grants AI057083 and AI102724 (to TES) and the Pittsburgh AIDS Research Training Program (T32 AI065380 to JAM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The Src family of non-receptor protein-tyrosine kinases is the largest tyrosine kinase family in the human kinome, with multiple members expressed in virtually every cell type. Three Src-family kinases (SFKs), c-Src, Fyn, and c-Yes, are found in most cell types while the remaining members have more restricted expression patterns, primarily in hematopoietic cell lineages [1]. SFKs regulate multiple cellular processes, including proliferation, survival, differentiation, adhesion and migration [2]. Strict control of SFK activity is essential to normal cellular and tissue homeostasis, while deregulation of SFK activity contributes to malignant transformation. Increased c-Src expression and activity are both observed in many forms of cancer and contribute to tumor cell proliferation, migration, and invasiveness [3]. All SFKs share a high degree of amino acid sequence homology and identical domain organization. The N-terminus of each family member encodes a signal sequence for myristoylation, a lipid modification critical to membrane localization. The myristoylation site is followed by a relatively short unique domain, which is the only non-homologous region across the kinase family. Most SFKs are also palmitoylated within this region, which contributes to membrane targeting [4]. Following the unique region is an SH3 domain, which binds proline-rich sequences that adopt a polyproline type-II (PPII) helical structure, an SH2 domain, which binds phosphotyrosine-containing sequences, an SH2-kinase linker, a bilobed kinase domain, and a C-terminal negative regulatory tail. All SFKs contain two conserved tyrosine phosphorylation sites important for regulation of kinase activity, which serve opposing roles [5]. Phosphorylation of Tyr416 (all residue numbering as per the structure of c-Src; PDB code 2SRC) in the activation loop stabilizes the active conformation of the kinase domain. Conversely, phosphorylation of Tyr527 in the C-terminal tail is required for downregulation of kinase activity. This site is phosphorylated by the separate (...truncated)


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Jamie A. Moroco, Jodi K. Craigo, Roxana E. Iacob, Thomas E. Wales, John R. Engen, Thomas E. Smithgall. Differential Sensitivity of Src-Family Kinases to Activation by SH3 Domain Displacement, PLOS ONE, 2014, Volume 9, Issue 8, DOI: 10.1371/journal.pone.0105629