Tyr394 and Tyr505 are Autophosphorylated in Recombinant Lck Protein‐tyrosine Kinase Expressed in <i>Escherichia coli</i>

Jullien, Pascale; Bougeret, Cécile; Camoin, Luc; Bodéus, Monique; Durand, H; Disanto, James P.; Fischer, Siegmund; Bénarous, Richard

doi:10.1111/j.1432-1033.1994.00589.x

Cited by 21 publications

(25 citation statements)

References 44 publications

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“…This result further demonstrates that the lack of time-dependent complete inactivation of Yes Ya by Csk is the result of autophosphorylation. Furthermore, these results indicate that the autophosphorylation that blocks Csk inactivation is on the main autophosphorylation site within the activation loop of Yes and Src, although other sites have been reported to also undergo autophosphorylation in the Src family (Osusky et al, 1995;Barker et al, 1995;Jullien et al, 1994). This result also indicates that autophosphorylation will likely have the same function in modulating Csk regulation of other PTKs in the Src family.…”

Section: Resultsmentioning

confidence: 74%

Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation

1998

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Csk phosphorylates Src family protein tyrosine kinases on a tyrosine residue near their C-terminus and downregulates their activity. We previously observed that this regulation requires a stoichiometric ratio of Csk : Src in a time-independent manner. In this report we examined this unusual kinetic behavior and found it to be caused by Src autophosphorylation. First, pre-incubation of Src with ATP-Mg led to time-dependent autophosphorylation of Src, activation of its kinase activity and loss of its ability to be inactivated by Csk. However, the autophosphorylated Src can still be phosphorylated by Csk. The SH2 binding site for phospho-Tyr of this hyperactive and doubly phosphorylated form of Src is not accessible. Second, dephosphorylation of autophosphorylated Src by protein tyrosine phosphatase 1B allowed Src to be inactivated by Csk. Third, protein tyrosine phosphatase 1B preferentially dephosphorylates the Src autophosphorylation site and allows for Src regulation by Csk. Finally, Yes, another member of the Src family, was also only partially inactivated when a sub-stoichiometric amount of Csk was used. Mutation of the tyrosine autophosphorylation site of Yes to a phenylalanine resulted in a mutant Yes enzyme that can be fully inactivated by a sub-stoichiometric amount of Csk in a time-dependent manner. These results demonstrate that Csk phosphorylation inactivates Src and Yes only when they are not previously autophosphorylated and Src autophosphorylation can block the inactivation by Csk phosphorylation. This conclusion suggests a dynamic model for the regulation of the Src family protein tyrosine kinases, which is discussed in the context of previously reported observations on the regulation of Src family protein tyrosine kinases.

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Section: Resultsmentioning

confidence: 74%

Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation

1998

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“…Autophosphorylation of Lck occurs primarily via an intermolecular trans-phosphorylation mechanism in vivo when expressed in bacterial systems (Jullien et al, 1994 (Figure 3c). The dierences in Lck activity in vivo do not result from a loss of intrinsic kinase activity as all the mutants are kinase active using enolase as a substrate in vitro (Figure 3d) Veillette, 1990;Amrein and Sefton, 1988;Gervais et al, 1993;Weil and Veillette, 1994).…”

Section: Mutations Immediately Downstream Of Y 394 Decrease Kinase Acmentioning

confidence: 99%

The activation loop in Lck regulates oncogenic potential by inhibiting basal kinase activity and restricting substrate specificity

2000

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The activities of Src-family non-receptor tyrosine kinases are regulated by structural changes that alter the orientation of key residues within the catalytic domain. In this study, we investigate the eects of activation loop mutations on regulation of the lymphocyte-speci®c kinase Lck (p56 lck ). Substitution of 5 ± 7 residues amino terminal to the conserved activation loop tyrosine (Y 394 ) increases kinase activity and oncogenic potential regardless of regulatory C-terminal tail phosphorylation levels (Y 505 ), while most mutations in the 13 residues carboxyl to Y 394 decrease kinase activity. Phosphorylation of the Cterminal regulatory tail is carried out by the cytosolic tyrosine kinase Csk and we ®nd that mutations upstream or downstream of Y394 or mutation of Y 394 do not aect the level of Y 505 phosphorylation. In addition, we report that mutations on either side of Y 394 aect substrate speci®city in vivo. We conclude that the high degree of conservation across the entire activation loop of Srcfamily kinases is critical for normal regulation of kinase activity and oncogenicity as well as substrate selection.

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“…Doubly phosphorylated c-Fgr is still active and can be partially inactivated by a phosphatase that selectively removes the phosphate from Y400 [23], consistent with an alternative mechanism of inactivation not relying on CSK. Lyn autophosphorylates tyrosine at its C-terminus, even in the absence of any effectors (Donella-Deana, A,, unpublished results) and the autophosphorylation of c-Src [24] and Lck [25] at their negative regulation sites has been also shown to occur. Moreover, autophosphorylation has been suggested as a possible mechanism for the C-terminal tyrosine phosphorylation of c-Src mutants not susceptible to phosphorylation by CSK [26].…”

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confidence: 99%

Sequence Specificity of C‐Terminal Src Kinase (Csk)

Ruzzene

Songyang

Marin

et al. 1997

European Journal of Biochemistry

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An eicosapeptide encompassing the C-terminal tail of c-Src (Tyr527) which is conserved in most Srcrelated protein kinases, is phosphorylated by C-terminal Src kinase (CSK) and by the two Src-related protein kinases c-Fgr and Lyn, with similar kinetic constants. Two related peptides reproducing the Cterminal segments of c-Src mutants defective in CSK phosphorylation [MacAuley, A., Okada, M., Nada, S., Nakagawa, H. & Cooper, J. A. (1993) Oncogene 8,117-1241 AFLEDSCTGTEPLYQBGENL (mutant number 28) and AFLEDIJITGTKPQYHPGENL (mutant number 29), proved a better and a much worse substrates, respectively than the wild-type peptide, with either CSK or the two Src kinases. By changing individual residues in the best peptide substrate, it was shown that the main element responsible for its improved phosphorylation is leucine at position -1 (instead of glutamine), while lysine at position -3 (instead of glutamate) has a detrimental effect, possibly accounting for the negligible phosphorylation of peptide derived from mutant number 29. By contrast to most peptide substrates, including the Src C-terminal peptides, which exhibit relatively high K,, values, a polyoma-virus-middle-T-antigen-(mT)-derived peptide with tyrosine embedded in a highly hydrophobic sequence (EEEPQFEEIPIYLELLP) exhibits with CSK a quite low K, value (63 pM). Consistent with this, the optimal sequence selected by CSK in an oriented peptide library is XXXIYMFFF. This is different from sequences selected by Lyn (DEEIYEELX) and c-Fgr (XEEIYGIFF), although they all share a high selection for a hydrophobic residue at n-1. In sharp contrast, T P K I I B I P~~"~, related to the catalytic domain of p72'kk, selects acidic residues at nearly all positions, n-1 included.These data support the notion that the features determining the specific phosphorylation of the Cterminal tyrosine residue of Src do not reside in the primary structure surrounding the target tyrosine. They also show that this site does not entirely fulfil the optimal consensus sequence recognized by CSK, disclosing the possibility that as yet unrecognized CSK targets structurally unrelated to the C-terminal tyrosine residue of Src kinases may exist.Keywords: protein-tyrosine kinase; C-terminal Src kinase ; c-Fgr ; Lyn; specificity.The C-terminal Src kinase (CSK) is a cytoplasmic protein tyrosine kinase which specifically phosphorylates Src-family kinases at their C-terminal tyrosine residue (equivalent to Tyr527 in c-Src), which is a down-regulation site [I, 21. CSK is, therefore, responsible for inhibiting the Tyr kinase activity of members of the Src family by inducing a locked conformation that results from the intramolecular interaction between the C-terminal tyrosine residue phosphorylated by CSK and the Src homology 2 (SH2) domain of the Src kinase 13-61, CSK is expressed in most tissues, while a related kinase with high sequence similarity, recently cloned with different names, megacaryocyte-associated tyrosine kinase (MATK), Ntk, Ctk, Batk, Lsk and HYL, [7 -121 displays a more restricted exp...

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Tyr394 and Tyr505 are Autophosphorylated in Recombinant Lck Protein‐tyrosine Kinase Expressed in Escherichia coli

Cited by 21 publications

References 44 publications

Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation

Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation

The activation loop in Lck regulates oncogenic potential by inhibiting basal kinase activity and restricting substrate specificity

Sequence Specificity of C‐Terminal Src Kinase (Csk)

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