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Tkl is the avian homolog of the mammalian lck tyrosine protein kinase gene.

ABSTRACT: We have tested the possibility that tkl, a partially characterized avian tyrosine protein kinase gene, is the chicken homolog of lck, a lymphocyte-specific mammalian gene. Using polymerase chain reactions, we have cloned sequences encoding the previously unidentified amino terminus of the tkl gene product. The newly defined unique domain of Tkl displayed significant identity (68%) to the equivalent region of the mammalian lck gene product, p56lck. This identity included a glycine residue at position 2 (present in all Scr-related tyrosine protein kinases) and a cysteine motif at positions 20 and 23, which allows binding of p56lck to CD4 and CD8 in mammalian T lymphocytes. A specific RNase protection assay revealed that, in contrast to a previous report (K. Strebhardt, J. I. Mullins, C. Bruck, and H. Rübsamen-Waigmann, Proc. Natl. Acad. Sci. USA 84:8778-8782, 1987), tkl expression is restricted to the lymphoid tissues thymus and spleen. Moreover, the absence of tkl transcripts in the bursa of Fabricius suggested that this gene is expressed in avian T lymphocytes but not in B lymphocytes. A polyclonal rabbit antiserum raised against the unique domain of Tkl recognized a 56-kDa polypeptide with associated protein kinase activity from avian thymus-derived cells. Additional studies showed that p56tkl is structurally similar to mammalian p56lck and that it is physically associated with the avian CD4 and CD8 T-cell surface antigens. It was also determined that tkl transcripts have one major type of 5' untranslated region (UTR), which differs greatly from the two known 5' UTRs of mammalian lck mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

SUBMITTER: Chow LM

PROVIDER: S-EPMC369554 | biostudies-other | 1992 Mar

REPOSITORIES: biostudies-other

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tkl is the avian homolog of the mammalian lck tyrosine protein kinase gene.

Chow L M LM Ratcliffe M J MJ Veillette A A

Molecular and cellular biology 19920301 3

We have tested the possibility that tkl, a partially characterized avian tyrosine protein kinase gene, is the chicken homolog of lck, a lymphocyte-specific mammalian gene. Using polymerase chain reactions, we have cloned sequences encoding the previously unidentified amino terminus of the tkl gene product. The newly defined unique domain of Tkl displayed significant identity (68%) to the equivalent region of the mammalian lck gene product, p56lck. This identity included a glycine residue at posi ...[more]

PMID: 1545804

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Project description:BackgroundUpon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood.MethodsKnock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies.ResultsCo-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45-/-/Csk-/- non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation.ConclusionsTaken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR.Major findingsOur data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation. Video Abstract.

Project description:Soluble extracts prepared from bovine thymus contain an angiotensin-I-phosphorylating activity that is activated several-fold by high concentrations of NaCl. Fractionation of this protein-tyrosine kinase activity by chromatography on DEAE-cellulose yields a major diffuse peak of activity. The enzymes responsible for this activity are found at much higher levels in extracts from bovine thymus than from bovine spleen. The peak of activity from the DEAE-cellulose column can be further separated into two major peaks by chromatography on heparin-agarose. The second peak to elute from the heparin-agarose column was previously purified through several chromatographic steps to yield a 40 kDa protein-tyrosine kinase (p40). We have now partially purified the early-eluting peak of kinase activity by chromatography on columns of butyl-agarose, protamine-agarose and Sephacryl S200. The enzyme was identified following covalent modification with 5'-fluorosulphonylbenzoyladenosine (FSBA) by reactivity with anti-FSBA antibodies. This procedure labelled a series of 52-56 kDa proteins. These proteins were also recognized by polyclonal anti-peptide antibodies raised against the C-terminal 33 amino acids of p56lck, a major T lymphocyte protein-tyrosine kinase. Peptide maps of the partially purified enzyme were identical to maps generated from p56lck obtained from LSTRA cells. These data suggest that bovine thymus p56lck is responsible for the activity found in the early-eluting peak from heparin-agarose. Antibodies raised against a peptide corresponding to amino acids 39-64 of p56lck, a sequence found near the N-terminus, recognized the slower-migrating, but not the faster-migrating, form of the enzyme, indicating that a fraction of the protein had been proteolysed near the N-terminus during purification. The partially purified bovine enzyme exhibited a restricted substrate specificity in vitro and did not readily phosphorylate human erythrocyte band 3, casein or histone, but was able to phosphorylate acid-treated enolase. The dilute enzyme present in fractions eluting from chromatography columns was unable to catalyse an autophosphorylation reaction. Autophosphorylation could be detected in more concentrated enzyme samples and was readily observed in immune-complex assays. The phosphorylation of angiotensin I by bovine thymus p56lck was weakly activated by polyionic compounds such as heparin and polylysine, and was strongly activated by high concentrations of NaCl.

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Tkl is the avian homolog of the mammalian lck tyrosine protein kinase gene.

Publications

tkl is the avian homolog of the mammalian lck tyrosine protein kinase gene.

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