Structural Basis for Domain–Domain Communication in a Protein Tyrosine Kinase, the C-terminal Src Kinase

Lin, Xiaofeng; Wang, Yuehao; Ahmadibeni, Yousef; Parang, Keykavous; Sun, Gongqin

doi:10.1016/j.jmb.2006.01.046

Cited by 30 publications

(44 citation statements)

References 27 publications

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“…This latter residue corresponds to F183 in Csk and to L255 in Src. Mutation of L255 in Src or F183 in Csk has been previously implicated in regulation of these kinases, providing further support for an important role for this position in Itk (35)(36)(37).…”

Section: Mutations In the Conserved Trp Pocket Deregulate Itk Activitymentioning

confidence: 68%

The Linker between SH2 and Kinase Domains Positively Regulates Catalysis of the Tec Family Kinases

2007

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Tec family nonreceptor tyrosine kinases are key immunological enzymes that control processes that range from T and B cell development to reorganization of the actin cytoskeleton. The full-length Tec kinases have been resistant to crystallization. This lack of structural data and the paucity of in vitro biochemical data for this kinase family leave a void in our understanding of Tec kinase regulation. In this report we have used interleukin-2 tyrosine kinase (Itk) as a model system to gain insight into the regulatory apparatus of the Tec kinases. Use of a quantitative in vitro kinase assay has uncovered an essential role for the short linker region flanked by the SH2 and kinase domains of Itk in positively regulating Itk catalytic activity. The precise residues that allosterically regulate Itk are conserved among Tec kinases, pointing to the conserved nature of this regulatory mechanism within the family. These findings indicate that Tec kinases are not regulated in the same manner as the Src kinases but rather share some of the regulatory features of Csk instead. ReceiVed December 5, 2006; ReVised Manuscript ReceiVed March 7, 2007 ABSTRACT: Tec family nonreceptor tyrosine kinases are key immunological enzymes that control processes that range from T and B cell development to reorganization of the actin cytoskeleton. The full-length Tec kinases have been resistant to crystallization. This lack of structural data and the paucity of in Vitro biochemical data for this kinase family leave a void in our understanding of Tec kinase regulation. In this report we have used interleukin-2 tyrosine kinase (Itk) as a model system to gain insight into the regulatory apparatus of the Tec kinases. Use of a quantitative in Vitro kinase assay has uncovered an essential role for the short linker region flanked by the SH2 and kinase domains of Itk in positively regulating Itk catalytic activity. The precise residues that allosterically regulate Itk are conserved among Tec kinases, pointing to the conserved nature of this regulatory mechanism within the family. These findings indicate that Tec kinases are not regulated in the same manner as the Src kinases but rather share some of the regulatory features of Csk instead.Protein kinases have evolved a variety of regulatory mechanisms that exploit the effects of site-specific phosphorylation and precise protein/domain interactions (1, 2). The largest family of nonreceptor protein tyrosine kinases consists of the eleven Src kinases (3) that each contain an N-terminal unique region, followed by an Src homology-3 (SH3 1 ) domain, Src homology-2 (SH2) domain, catalytic domain, and a C-terminal tail with a conserved regulatory tyrosine. A significant leap in our understanding of kinase regulation has been provided by the three-dimensional structures of two Src family kinases (Hck and Src) in their inactive states (4-6). These structures reveal that the SH3 and SH2 domains are located on the "back" surface of the kinase domain mediating a network of intramolecular interactions th...

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Section: Mutations In the Conserved Trp Pocket Deregulate Itk Activitymentioning

confidence: 68%

The Linker between SH2 and Kinase Domains Positively Regulates Catalysis of the Tec Family Kinases

2007

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“…In mammalian Csk, substitution of this residue with alanine led to a loss of kinase activity. 38 To test the importance of this residue in MvCsk, we introduced the Asn residue found in mammalian Csk. We produced a G236N mutant form of MvCsk, expressed and purified the GST fusion protein, and tested the activity against a synthetic substrate peptide.…”

Section: Resultsmentioning

confidence: 99%

Regulation of Src and Csk Nonreceptor Tyrosine Kinases in the Filasterean Ministeria vibrans

et al. 2014

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The development of the phosphotyrosine-based signaling system predated the evolution of multicellular animals. Single-celled choanoflagellates, the closest living relatives to metazoans, possess numerous tyrosine kinases, including Src family nonreceptor tyrosine kinases. Choanoflagellates also have Csk (C-terminal Src kinase), the enzyme that regulates Src in metazoans; however, choanoflagellate Csk kinases fail to repress the cognate Src. Here, we have cloned and characterized Src and Csk kinases from Ministeria vibrans, a filasterean (the sister group to metazoans and choanoflagellates). The two Src kinases (MvSrc1 and MvSrc2) are enzymatically active Src kinases, although they have low activity toward mammalian cellular proteins. Unexpectedly, MvSrc2 has significant Ser/Thr kinase activity. The Csk homologue (MvCsk) is enzymatically inactive and fails to repress MvSrc activity. We suggest that the low activity of MvCsk is due to sequences in the SH2—kinase interface, and we show that a point mutation in this region partially restores MvCsk activity. The inactivity of filasterean Csk kinases is consistent with a model in which the stringent regulation of Src family kinases arose more recently in evolution, after the split between choanoflagellates and multicellular animals.

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“…The autophosphorylation of c-Src characterizes a well described mechanism. Under nonactivating conditions, Src kinases are maintained in an autoinhibited state by intramolecular interactions of their SH2 and SH3 domains (45)(46)(47). In the inhibited configuration, SH3 binding to a polyproline sequence in the linker region between the SH2 and kinase domains regulates the inactive conformation of the catalytic lobes.…”

Section: Discussionmentioning

confidence: 99%

Presenilin 1 Affects Focal Adhesion Site Formation and Cell Force Generation via c-Src Transcriptional and Posttranslational Regulation

Waschbüsch

Born

Niediek

et al. 2009

Journal of Biological Chemistry

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Presenilin 1 and 2 (PS) are critical components of the ␥-secretase complex that cleaves type I transmembrane proteins within their transmembrane domains. This process leads to release of proteolytically processed products from cellular membranes and plays an essential role in signal transduction or vital functions as cell adhesion. Here we studied the function of presenilins in cell-matrix interaction of wild-type and PS knock-out mouse embryonic fibroblasts. We found for PS1؊/؊ cells an altered morphology with significantly reduced sizes of focal adhesion sites compared with wild type. Cell force analyses on micropatterned elastomer films revealed PS1 ؊/؊ cell forces to be reduced by 50%. Pharmacological inhibition confirmed this function of ␥-secretase in adhesion site and cell force formation. On the regulatory level, PS1 deficiency was associated with strongly decreased phosphotyrosine levels of focal adhesion site-specific proteins. The reduced tyrosine phosphorylation was caused by a down-regulation of c-Src kinase activity primarily at the level of c-Src transcription. The direct regulatory connection between PS1 and c-Src could be identified with ephrinB2 as PS1 target protein. Overexpression of ephrinB2 cytoplasmic domain resulted in its nuclear translocation with increased levels of c-Src and a full complementation of the PS1 ؊/؊ adhesion and phosphorylation phenotype. Cleavage of full-length EB2 and subsequent intracellular domain translocation depended on PS1 as these processes were only found in WT cells. Therefore, we conclude that ␥-secretase is vital for controlling cell adhesion and force formation by transcriptional regulation of c-Src via ephrinB2 cleavage. PS12 and PS2 are aspartyl proteases forming the active components of the ␥-secretase complex. This complex cleaves type 1 transmembrane proteins within their transmembrane domains (1-3). A prerequisite for ␥-secretase cleavage is shedding of the ectodomains of the respective transmembrane proteins close to their transmembrane domains (4, 5). Subsequently, cleavage of the remaining transmembrane protein stubs by ␥-secretase leads to the release of the two cleavage products from the membrane (2). The freed products of some proteins play important roles in different signaling pathways e.g. in cell-cell adhesion or cell differentiation (1, 2, 6). ␥-Secretase also cleaves the amyloid precursor protein, thereby producing the neurotoxic amyloid ␤-peptide that precipitates in amyloid plaques in Alzheimer disease (1, 7). Another example can be found in notch signaling, which is essential for embryonal development. PS-dependent cleavage of notch leads to release of the so called notch intracellular domain. This domain translocates to the nucleus where it acts as a transcriptional coactivator (2). In vivo studies revealed strong phenotypes for PS1 Ϫ/Ϫ and even more for PS1 Ϫ/Ϫ PS2 Ϫ/Ϫ double knock-out mice that closely resemble notch mutant mice, whereas PS2 Ϫ/Ϫ mice are barely affected (8). These observations argue for the ability of PS1 to complemen...

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Structural Basis for Domain–Domain Communication in a Protein Tyrosine Kinase, the C-terminal Src Kinase

Cited by 30 publications

References 27 publications

The Linker between SH2 and Kinase Domains Positively Regulates Catalysis of the Tec Family Kinases

The Linker between SH2 and Kinase Domains Positively Regulates Catalysis of the Tec Family Kinases

Regulation of Src and Csk Nonreceptor Tyrosine Kinases in the Filasterean Ministeria vibrans

Presenilin 1 Affects Focal Adhesion Site Formation and Cell Force Generation via c-Src Transcriptional and Posttranslational Regulation

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