Sequence Specificity of C‐Terminal Src Kinase (Csk)

Ruzzene, Maria; Songyang, Zhou; Marin, Oriano; Donella‐Deana, Arianna; Brunati, Anna Maria; Guerra, Bárbara; Agostinis, Patrizia; Cantley, Lewis C.; Pinna, Lorenzo A.

doi:10.1111/j.1432-1033.1997.t01-1-00433.x

Cited by 32 publications

(33 citation statements)

References 45 publications

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“…The Cterminal dCSK phosphorylation site is conserved in other LATS homologs, suggesting that mammalian LATS molecules may also be substrates of CSK. Interestingly, Pinna and colleagues have defined the optimal peptide substrates for mammalian CSK (Ruzzene et al, 1997). This predicts that the C-terminal tyrosine motifs of the mammalian LATS molecules would be better CSK substrates than Src.…”

Section: Dcsk and Lats Function Together In Size Controlmentioning

confidence: 99%

A genetic screen for modifiers of the lats tumor suppressor gene identifies C-terminal Src kinase as a regulator of cell proliferation in Drosophila

Stewart

Huang

et al. 2003

Oncogene

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Disrupting mechanisms that control cell proliferation, cell size and apoptosis can cause changes in animal and tissue size and contribute to diseases such as cancer. The LATS family of serine/threonine kinases control tissue size by regulating cell proliferation and function as tumor suppressor genes in both Drosophila and mammals. In order to understand the role of lats in size regulation, we performed a genetic modifier screen in Drosophila to identify components of the lats signaling pathway. Mutations in the Drosophila homolog of C-terminal Src kinase (dcsk) were identified as dominant modifiers of both lats gain-of-function and loss-of-function phenotypes. Homozygous dcsk mutants have enlarged tissue phenotypes similar to lats and FACS and immunohistochemistry analysis of these tissues revealed that dcsk also regulates cell proliferation during development. Animals having mutations in both dcsk and lats display cell overproliferation phenotypes more severe than either mutant alone, demonstrating these genes function together in vivo to regulate cell numbers. Furthermore, homozygous dcsk phenotypes can be partially suppressed by overexpression of lats, indicating that lats is a downstream mediator of dcsk function in vivo. Finally, we show that dCSK phosphorylates LATS in vitro at a conserved C-terminal tyrosine residue, which is critical for normal LATS function in vivo. Taken together, these results demonstrate a role for dCSK in regulating cell numbers during development by inhibiting cell proliferation and suggest that lats is one of the mediators of the dcsk phenotype.

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Section: Dcsk and Lats Function Together In Size Controlmentioning

confidence: 99%

A genetic screen for modifiers of the lats tumor suppressor gene identifies C-terminal Src kinase as a regulator of cell proliferation in Drosophila

Stewart

Huang

et al. 2003

Oncogene

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“…Early efforts to understand Csk substrate specificity used peptides mimicking this phosphorylation site (8,16). However, such peptides are Ϸ1,000 times less efficient than SFKs as substrates for Csk (8,16,17).…”

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confidence: 99%

“…However, such peptides are Ϸ1,000 times less efficient than SFKs as substrates for Csk (8,16,17). By screening a random peptide library, Cole and coworkers (17) identified an optimal peptide substrate for Csk with the sequence of EEEIYFFF.…”

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confidence: 99%

Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase

Lee

Lin

Nam

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Protein tyrosine kinases (PTK) are key enzymes of mammalian signal transduction. For the fidelity of signal transduction, each PTK phosphorylates only one or a few proteins on specific Tyr residues. Substrate specificity is thought to be mediated by PTKsubstrate docking interactions and recognition of the phosphorylation site sequence by the kinase active site. However, a substratedocking site has not been determined on any PTK. C-terminal Src kinase (Csk) is a PTK that specifically phosphorylates Src family kinases on a C-terminal Tyr. In this study, by sequence alignment and site-specific mutagenesis, we located a substrate-docking site on Csk. Mutations in the docking site disabled Csk to phosphorylate, regulate, and complex with Src but only moderately affected its general kinase activity. A peptide mimicking the docking site potently inhibited (IC50 ‫؍‬ 21 M) Csk phosphorylation of Src but only moderately inhibited (IC50 ‫؍‬ 422 M) its general kinase activity. Determination of the substrate-docking site provides the structural basis of substrate specificity in Csk and a model for understanding substrate specificity in other PTKs.

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“…The sequence of the phosphoacceptor site (Tyr-222) is in fact markedly divergent from the optimal sequences selected by c-Fgr and Lyn in an oriented peptide library (20), and it lacks, in particular, the crucial hydrophobic residue at position n-1, which is important for site recognition by all Src kinases (20,21). Indeed unphosphorylated HS1 and its truncated forms are not appreciably phosphorylated by c-Fgr (Fig.…”

Section: ]Atp and Increasing Concentrations Of Either The Recombinantmentioning

confidence: 98%

Molecular Features Underlying the Sequential Phosphorylation of HS1 Protein and Its Association with c-Fgr Protein-tyrosine Kinase

Brunati¹,

Donella‐Deana²,

James³

et al. 1999

Journal of Biological Chemistry

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The hematopoietic lineage cell-specific protein HS1 was shown to undergo a process of sequential phosphorylation both in vitro and in vivo, which is synergistically mediated by Syk and Src family protein-tyrosine kinases and essential for B cell antigen receptor-mediated apoptosis. We have now identified tyrosine 222 as the HS1 residue phosphorylated by the Src family protein kinases c-Fgr and Lyn, and we show that a truncated form of HS1 (HS1-208-401) lacking the N-terminal putative DNA binding region and the C-terminal Src homology 3 (SH3) domain is still able to undergo all the steps of sequential phosphorylation as efficiently as fulllength HS1. We also show that a stable association of phospho-HS1 with c-Fgr through its SH2 domain requires previous autophosphorylation of the kinase and is prevented by subsequent phosphorylation of Tyr-222. Kinetic studies with HS1 and its truncated forms previously phosphorylated by Syk and with a peptide substrate reproducing the sequence around tyrosine 222 support the view that efficient phosphorylation of HS1 by Src family protein kinases entirely relies on TyrP-SH2 domain interaction with negligible, if any, contribution of local specificity determinants. Our data indicate that the proline-rich region of HS1 bordered by tyrosyl residues affected by Syk and Src family kinases represents a functional domain designed to undergo a process of sequential phosphorylation. HS11 is an intracellular protein with expression that is limited to hematopoietic and lymphoid cells (1). The gene sequence shows that is composed of 486 amino acids with a predicted M r of ϳ54 kDa. In contrast, HS1 has been constantly isolated as a protein with an apparent M r of ϳ75 kDa, as judged from SDS-PAGE, giving rise to the suggestion that it might be heavily affected by post-translational modifications, e.g. glycosylation (1). The amino acid sequence of HS1 contains a variety of structurally significant motifs, including an N-terminal region responsible for the binding of mitochondrial protein HS1-associated protein X-1 (2), followed by four 37-amino acids repeats that can form a helix-turn-helix structure frequently found in the DNA binding domain of various transcriptional factors (1); this region contains three putative phosphatidylinositol 4,5-bisphosphate binding motifs and has been suggested to be involved in F-actin binding (3), a proline-rich region localized to the C-terminal central moiety, which may represent an SH3 binding motif and is ending with a stretch of proline-glutamate repeats, and an SH3 domain located at the C-terminal extremity (see schematic representation of HS1 in Fig. 1).Evidence that HS1 plays a role in the receptor-mediated apoptosis and proliferative responses was provided by the analysis of HS1-deficient mice (4) and WEH1-231 B lymphoma cells (5-7). The observation that HS1 is readily tyrosine phosphorylated after B cell antigen receptor cross-linking, in parallel with activation of Src family kinases (8, 9), is also consistent with a role for HS1 in signal transduct...

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Sequence Specificity of C‐Terminal Src Kinase (Csk)

Cited by 32 publications

References 45 publications

A genetic screen for modifiers of the lats tumor suppressor gene identifies C-terminal Src kinase as a regulator of cell proliferation in Drosophila

A genetic screen for modifiers of the lats tumor suppressor gene identifies C-terminal Src kinase as a regulator of cell proliferation in Drosophila

Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase

Molecular Features Underlying the Sequential Phosphorylation of HS1 Protein and Its Association with c-Fgr Protein-tyrosine Kinase

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