Synthesis, purification, and characterization of the cytoplasmic domain of the human insulin receptor using a baculovirus expression system

The cytoplasmic domain of the beta subunit of the human insulin receptor has been overexpressed in insect cells using the baculovirus expression system. A recombinant baculovirus (BIR-2) was constructed by inserting the human insulin proreceptor cDNA fragment that encodes the cytoplasmic domain of the receptor into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Synthesis of the protein (baculovirus insulin receptor kinase (BIRK), Mr 48,000) in BIR-2-infected Spodoptera frugiperda (Sf9) cells was detected 24 h after infection and maximal accumulation (2-3% of the cytosolic protein) was achieved 48-72 h post-infection. The expressed protein is active as a soluble protein tyrosine kinase, both in Sf9 cells and in vitro. Rapid purification to near homogeneity was accomplished by sequential chromatography on Fast-Q-Sepharose and phenyl-Superose with an overall yield of 35% and a specific activity with histone as substrate of 20 nmol/min/mg protein. Autophosphorylation activated the intrinsic kinase activity of BIRK and decreased its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using a combination of tryptic digestion and immunoprecipitation with specific antipeptide antisera, it was ascertained that 30-40% of the 32P incorporated into BIRK by autophosphorylation is in the carboxyl-terminal domain (that includes tyrosyl residues 1316 and 1322 of the human proreceptor). Of the remaining radioactivity, 75% is in the amino-terminal domain (that includes tyrosyl residues 953, 960, 972, 999, and 1075) and 25% is in the conserved autophosphorylation domain (including tyrosyl residues 1146, 1150, and 1151). Limited digestion of BIRK with trypsin yielded a fragment, Mr 38,000, that lacks the carboxyl-terminal domain. This fragment exhibits protein tyrosine kinase activity that is stimulated by autophosphorylation. The properties of the soluble, monomeric BIRK are similar to those of the intact, activated, oligomeric insulin receptor kinase with respect to specificity, immunoreactivity, divalent cation requirements, and specific activity. These observations coupled with the ease of producing 0.4 mg of purified enzyme from 100 ml of suspension culture suggest that BIRK will be useful for biochemical and biophysical analysis of the insulin receptor protein tyrosine kinase.     

Two-dimensional phosphopeptide analysis of the autophosphorylation cascade of a soluble insulin receptor tyrosine kinase. The tyrosines phosphorylated are typical of those observed following phosphorylation of the heterotetrameric insulin receptor in intact cells.

A soluble derivative of the human insulin receptor cytoplasmic domain, as expressed in insect cells via a Baculovirus vector, is an active protein-tyrosine kinase. In the present study, we find that three forms of the enzyme (48, 43, and 38 kDa) can be partially purified by MonoQ fast protein liquid chromatography. Two-dimensional thin layer phosphopeptide mapping reveals that the 48-kDa enzyme undergoes a rapid autophosphorylation on the same tyrosines (residues 1158, 1162, 1163, 1328, and 1334) that have previously been shown to be major autophosphorylation sites on the native insulin receptor beta-subunit in intact cells. Furthermore, the 48- and 43-kDa proteins are phosphorylated on serine residues by a serine kinase(s) that copurifies through MonoQ fast protein liquid chromatography. Tyrosine autophosphorylation sites 1328 and 1334 and virtually all serine phosphorylation sites are absent in the 38-kDa kinase. Partial tryptic proteolysis of the 48-kDa kinase generates a core 38-kDa enzyme that undergoes autophosphorylation almost exclusively on tyrosines 1158, 1162, and 1163. Phosphorylation of these tyrosine residues occurs in a cascade manner analogous to that found in the intact insulin receptor beta-subunit.     

Activation of the purified protein tyrosine kinase domain of the epidermal growth factor receptor

Biological responses to epidermal growth factor (EGF) depend on the ligand-stimulated protein tyrosine kinase activity of its receptor. To further characterize the enzymatic activity of the EGF receptor, the baculovirus expression system was used to express the cytoplasmic protein tyrosine kinase domain of the EGF receptor. Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus correctly expressed an active tyrosine kinase domain of the EGF receptor as demonstrated by 35S metabolic labeling, immunoblotting with anti-EGF receptor and anti-phosphotyrosine antibodies, and autophosphorylation analysis. The kinase domain (Mr 66,000) was purified to near homogeneity using a monoclonal anti-phosphotyrosine antibody column, providing 0.5 mg of kinase domain/liter of Sf9 cells (23% yield). The purified kinase domain exhibited a strong preference for Mn2+ compared to Mg2+. The specific activity of the kinase domain was low compared to purified, EGF-activated EGF receptor. However, the addition of sphingosine or ammonium sulfate greatly increased the activity of the kinase domain to equal or exceed the activity of ligand-activated holo EGF receptor. These results indicate that the addition of sphingosine or ammonium sulfate to the purified kinase domain can mimic the effect of EGF to induce a conformation of the holo EGF receptor which is optimal for tyrosine kinase activity. Deletion of the ligand binding domain, analogous to that which occurs in erb B, is not sufficient to fully activate the kinase, implying that EGF causes conformational changes additional to removal of an inhibitory constraint.     

cDNA cloning and characterization of eck, an epithelial cell receptor protein-tyrosine kinase in the eph/elk family of protein kinases.

A human epithelial (HeLa) cDNA library was screened with degenerate oligonucleotides designed to hybridize to highly conserved regions of protein-tyrosine kinases. One cDNA from this screen was shown to contain a putative protein-tyrosine kinase catalytic domain and subsequently used to isolate another cDNA from a human keratinocyte library that encompasses the entire coding region of a 976-amino-acid polypeptide. The predicted protein has an external domain of 534 amino acids with a presumptive N-terminal signal peptide, a transmembrane domain, and a cytoplasmic domain of 418 amino acids that includes a canonical protein-tyrosine kinase catalytic domain. Molecular phylogeny indicates that this protein kinase is closely related to eph and elk and that this receptor family is more closely related to the non-receptor protein-tyrosine kinase families than to other receptor protein-tyrosine kinases. Antibodies raised against a TrpE fusion protein immunoprecipitated a 130-kDa protein that became phosphorylated on tyrosine in immune complex kinase assays, indicating that this protein is a bona fide protein-tyrosine kinase. Analysis of RNA from 13 adult rat organs showed that the eck gene is expressed most highly in tissues that contain a high proportion of epithelial cells, e.g., skin, intestine, lung, and ovary. Several cell lines of epithelial origin were found to express the eck protein kinase at the protein and RNA levels. Immunohistochemical analysis of several rat organs also showed staining in epithelial cells. These observations prompted us to name this protein kinase eck, for epithelial cell kinase.     

H(2)O(2)-induced tyrosine phosphorylation of protein kinase cdelta by a mechanism independent of inhibition of protein-tyrosine phosphatase in CHO and COS-7 cells

It has been proposed that H(2)O(2) increases tyrosine phosphorylation of cellular proteins by inhibiting protein-tyrosine phosphatase through oxidation of the cysteine residue of the enzyme essential for its catalytic activity. Tyrosine phosphorylation of the delta isoform of protein kinase C (PKC) was induced by H(2)O(2) in CHO and COS-7 cells. H(2)O(2) also induced activation of mitogen-activated protein kinase. Vanadate and molybdate, which inhibit protein-tyrosine phosphatase by binding to its active site, did not induce tyrosine phosphorylation of PKCdelta, but enhanced H(2)O(2)-induced tyrosine phosphorylation of PKCdelta in the cell. The oxoanions, however, generated the active form of mitogen-activated protein kinase. Another protein-tyrosine phosphatase inhibitor, phenylarsine oxide, which bridges the thiol residues of the enzyme, induced tyrosine phosphorylation of PKCdelta, and the reaction was enhanced by vanadate. These results suggest that inhibition of protein-tyrosine phosphatase is insufficient for induction of tyrosine phosphorylation of PKCdelta in the cells, and that presumably activation of protein-tyrosine kinase may be essential for tyrosine phosphorylation of the PKC isoform.     

A strong protein-tyrosine kinase activity is associated with a baculovirus-expressed chicken tkl gene.

We have previously described a gene named tkl (tyrosine kinase related to lck). It belongs to the src family of protein-tyrosine kinases and among these it has significant homology to the lck gene (lymphoide cell kinase). The tkl gene product may represent the avian homolog of Lck, which is believed to participate in a lymphocyte-specific signal transduction pathway by association with a membrane receptor. To study the biochemical properties of the protein, a nearly complete tkl gene (isolated from a cDNA library from chicken spleen cells) was expressed in a baculovirus system. Approximately 10% of the extracted protein consisted of the soluble 51-kDa Tkl protein (p51tkl) at 40 h post-infection. This protein was found to be phosphorylated on tyrosine and serine residues at a ratio of 5:1. As expected, glycosylation or myristoylation could not be detected. Immunocomplex kinase assays indicated strong autophosphorylation of p51tkl at tyrosine residues and phosphorylation of exogenous substrates such as D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histones H2b and H4, and casein. This protein-tyrosine kinase activity also exhibited a marked preference for Mn2+ compared to Mg2+. The high level expression of enzymatically active Tkl should provide an excellent tool to further study the biological functions of this class of enzymes.     

Tyrosine phosphorylation of BCR by FPS/FES protein-tyrosine kinases induces association of BCR with GRB-2/SOS.

The human bcr gene encodes a protein with serine/threonine kinase activity, CDC24/dbl homology, a GAP domain, and an SH2-binding region. However, the precise physiological functions of BCR are unknown. Coexpression of BCR with the cytoplasmic protein-tyrosine kinase encoded by the c-fes proto-oncogene in Sf-9 cells resulted in stable BCR-FES protein complex formation and tyrosine phosphorylation of BCR. Association involves the SH2 domain of FES and a novel binding domain localized to the first 347 amino acids of the FES N-terminal region. Deletion of the homologous N-terminal BCR-binding domain from v-fps, a fes-related transforming oncogene, abolished transforming activity and tyrosine phosphorylation of BCR in vivo. Tyrosine phosphorylation of BCR in v-fps-transformed cells induced its association with GRB-2/SOS, the RAS guanine nucleotide exchange factor complex. These data provide evidence that BCR couples the cytoplasmic protein-tyrosine kinase and RAS signaling pathways.     

Identification of tyrosine 67 in bovine brain myelin basic protein as a specific phosphorylation site for thymus p56lck.

The protein-tyrosine kinase p56lck exhibits a restricted substrate specificity in vitro but can efficiently phosphorylate bovine myelin basic protein (MBP). Results obtained from both peptide mapping and fast atom bombardment mass spectrometry indicate that tyrosine 67 in the sequence -Thr-Thr-His-Tyr67-Gly-Ser-Leu-Pro-Gln-Lys- in bovine MBP is the specific phosphorylation site. p56lck does not phosphorylate the acidic, cytoplasmic domain of erythrocyte band 3. In contrast, p40, another protein-tyrosine kinase purified from bovine thymus that readily phosphorylates band 3, does not phosphorylate MBP. Therefore, MBP and band 3 may prove to be useful substrates for distinguishing between various tyrosine kinases on the basis of substrate specificity. In addition, identification of the recognition sequence in MBP for p56lck may contribute to an understanding of the structural features of physiological substrates for this kinase.     

Characterization of elk, a brain-specific receptor tyrosine kinase.

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extracellular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system.     

Phosphorylation sites in human erythrocyte band 3 protein

The human red cell anion-exchanger, band 3 protein, is one of the main phosphorylated proteins of the erythrocyte membrane. Previous studies from this laboratory have shown that ATP-depletion of the red blood cell decreased the anion-exchange rate, suggesting that band 3 protein phosphorylation could be involved in the regulation of anion transport function (Bursaux et al. (1984) Biochim. Biophys. Acta 777, 253-260). Phosphorylation occurs mainly on the cytoplasmic domain of the protein and the major site of phosphorylation was assigned to tyrosine-8 (Dekowski et al. (1983) J. Biol. Chem. 258, 2750-2753). This site being very far from the integral, anion-exchanger domain, the aim of the present study was to determine whether phosphorylation sites exist in the integral domain. The phosphorylation reaction was carried out on isolated membranes in the presence of [gamma-32P]ATP and phosphorylated band 3 protein was then isolated. Both the cytoplasmic and the membrane spanning domains were purified. The predominant phosphorylation sites were found on the cytoplasmic domain. RP-HPLC analyses of the tryptic peptides of whole band 3 protein, and of the isolated cytoplasmic and membrane-spanning domains allowed for the precise localization of the phosphorylated residues. 80% of the label was found in the N-terminal tryptic peptide (T-1), (residues 1-56). In this region, all the residues susceptible to phosphorylation were labeled but in varying proportion. Under our conditions, the most active membrane kinase was a tyrosine kinase, activated preferentially by Mn2+ but also by Mg2+. Tyrosine-8 was the main phosphate acceptor residue (50-70%) of the protein, tyrosine-21 and tyrosine-46 residues were also phosphorylated but to a much lesser extent. The main targets of membrane casein kinase, preferentially activated by Mg2+, were serine-29, serine-50, and threonine(s)-39, -42, -44, -48, -49, -54 residue(s) located in the T-1 peptide. A tyrosine phosphatase activity was copurified with whole band 3 protein which dephosphorylates specifically P-Tyr-8, indicating a highly exchangeable phosphate. The membrane-spanning fragment was only faintly labeled.     

Src protein-tyrosine kinase structure and regulation

Src and Src-family protein kinases are proto-oncogenes that play key roles in cell morphology, motility, proliferation, and survival. v-Src (a viral protein) is encoded by the chicken oncogene of Rous sarcoma virus, and Src (the cellular homologue) is encoded by a physiological gene, the first of the proto-oncogenes. From the N- to C-terminus, Src contains an N-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a C-terminal regulatory tail. The chief phosphorylation sites of Src include tyrosine 416 that results in activation from autophosphorylation and tyrosine 527 that results in inhibition from phosphorylation by C-terminal Src kinase. In the restrained state, the SH2 domain forms a salt bridge with phosphotyrosine 527, and the SH3 domain binds to the kinase domain via a polyproline type II left-handed helix. The SH2 and SH3 domains occur on the backside of the kinase domain away from the active site where they stabilize a dormant enzyme conformation. Protein-tyrosine phosphatases such as PTPalpha displace phosphotyrosine 527 from the Src SH2 domain and mediate its dephosphorylation leading to Src kinase activation. C-terminal Src kinase consists of an SH3, SH2, and kinase domain; it lacks an N-terminal myristoyl group and a C-terminal regulatory tail. Its X-ray structure has been determined, and the SH2 lobe occupies a position that is entirely different from that of Src. Unlike Src, the C-terminal Src kinase SH2 and SH3 domains stabilize an active enzyme conformation. Amino acid residues in the alphaD helix near the catalytic loop in the large lobe of C-terminal Src kinase serve as a docking site for the physiological substrate (Src) but not for an artificial substrate (polyGlu(4)Tyr).     

A soluble insulin receptor kinase catalyzes ordered phosphorylation at multiple tyrosines of dodecapeptide substrates.

At present, the requirements for efficient phosphorylation of exogenous substrates by protein-tyrosine kinases are largely unknown. The proton resonances of each of the 3 tyrosines of the dodecapeptide substrate RRDIYETDYYRK are well resolved in the aromatic region of the 1H NMR spectra: thus, it is feasible to directly monitor phosphorylation at each site. A soluble approximately 48-kDa derivative of the human insulin receptor cytoplasmic protein-tyrosine kinase domain phosphorylates this peptide at all 3 tyrosine sites and does so in a highly ordered and progressive manner (Y9, then Y10 and finally Y5), proceeding to full stoichiometry at each site before phosphorylating the next. This experimental system now provides an approach by which to follow the stereochemical requirements and dynamics of substrate phosphorylation by a protein-tyrosine kinase in solution in real time.     

A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps.

Proteins encoded by oncogenes such as v-fps/fes, v-src, v-yes, v-abl, and v-fgr are cytoplasmic protein tyrosine kinases which, unlike transmembrane receptors, are localized to the inside of the cell. These proteins possess two contiguous regions of sequence identity: a C-terminal catalytic domain of 260 residues with homology to other tyrosine-specific and serine-threonine-specific protein kinases, and a unique domain of approximately 100 residues which is located N terminal to the kinase region and is absent from kinases that span the plasma membrane. In-frame linker insertion mutations in Fujinami avian sarcoma virus which introduced dipeptide insertions into the most stringently conserved segment of this N-terminal domain in P130gag-fps impaired the ability of Fujinami avian sarcoma virus to transform rat-2 cells. The P130gag-fps proteins encoded by these transformation-defective mutants were deficient in protein-tyrosine kinase activity in rat cells. However v-fps polypeptides derived from the mutant Fujinami avian sarcoma virus genomes and expressed in Escherichia coli as trpE-v-fps fusion proteins displayed essentially wild-type enzymatic activity, even though they contained the mutated sites. Deletion of the N-terminal domain from wild-type and mutant v-fps bacterial proteins had little effect on autophosphorylating activity. The conserved N-terminal domain of P130gag-fps is therefore not required for catalytic activity, but can profoundly influence the adjacent kinase region. The presence of this noncatalytic domain in all known cytoplasmic tyrosine kinases of higher and lower eucaryotes argues for an important biological function. The relative inactivity of the mutant proteins in rat-2 cells compared with bacteria suggests that the noncatalytic domain may direct specific interactions of the enzymatic region with cellular components that regulate or mediate tyrosine kinase function.     

Cloning and characterization of Dfak56, a homolog of focal adhesion kinase, in Drosophila melanogaster.

The focal adhesion kinase (FAK) protein-tyrosine kinase plays important roles in cell adhesion in vertebrates. Using polymerase chain reaction-based cloning strategy, we cloned a Drosophila gene that is homologous to the vertebrate FAK family of protein-tyrosine kinases. We designated this gene Dfak56 and characterized its gene product. The overall protein structure and deduced amino acid sequence of Dfak56 show significant similarity to those of FAK and PYK2. Dfak56 has in vitro autophosphorylation activity at tyrosine residues. Expression of the Dfak56 mRNA and the protein was observed in the central nervous system and the muscle-epidermis attachment site in the embryo, where Drosophila position-specific integrins are localized. The results suggest that like FAK in vertebrates, Dfak56 functions downstream of integrins. Dfak56 was tyrosine-phosphorylated upon integrin-dependent attachment of the cell to the extracellular matrix. We conclude that the Dfak56 tyrosine kinase is involved in integrin-mediated cell adhesion signaling and thus is a functional homolog of vertebrate FAK.     

Expression and purification of the intact cytoplasmic domain of the human ephrin receptor A2 tyrosine kinase in Escherichia coli

The ephrin receptor A2 (EphA2) is an integral membrane protein tyrosine kinase and a member of the Eph family, the largest known family of receptor tyrosine kinases. EphA2 overexpression is sufficient to transform normal epithelial cells into an aggressive, metastatic phenotype. In normal cells, EphA2 negatively regulates cell growth and invasiveness. Here we report expression of the intact cytoplasmic domain (juxtamembrane linker, tyrosine kinase, and sterile alpha motif domains) of the human EphA2 receptor in an Escherichia coli system. The expressed protein was purified to near homogeneity by use of metal chelation chromatography combined with removal of vector-encoded tags by specific proteolysis. The cytoplasmic domains of EphA2 are expressed as an active kinase, with the expressed protein found to contain phosphorylated tyrosine residues. In addition, protein tyrosine phosphorylation appears only after EphA2 expression is induced and is removable with alkaline phosphatase treatment. The enzyme was purified 5-fold in yields that average 10-30 mg/L of active EphA2 cytoplasmic domains, which will now be used for further biophysical and structural characterization.     

A major site of tyrosine phosphorylation within the SH2 domain of Fujinami sarcoma virus P130gag-fps is not required for protein-tyrosine kinase activity or transforming potential.

Phosphorylation of the major autophosphorylation site (Tyr-1073) within Fujinami sarcoma virus P130gag-fps activates both the intrinsic protein-tyrosine kinase activity and transforming potential of the protein. In this report, a second site of autophosphorylation Tyr-836 was identified. This tyrosine residue is found within a noncatalytic domain (SH2) of P130gag-fps that is required for full protein-kinase activity in both rat and chicken cells. Autophosphorylation of this tyrosine residue implies that the SH2 region lies near the active site in the catalytic domain in the native protein and thus possibly regulates its enzymatic activity. Four mutations have occurred within the SH2 domain between the c-fps and v-fps proteins. Tyr-836 is one of these changes, being a Cys in c-fps. Site-directed mutagenesis was used to investigate the function of this autophosphorylation site. Substitution of Tyr-836 with a Phe had no apparent effect on the transforming ability or protein-tyrosine kinase activity of P130gag-fps in rat-2 cells. Mutagenesis of both autophosphorylation sites (Tyr-1073 and Tyr-836) did not reveal any cooperation between these two phosphorylation sites. The implications of the changes within the SH2 region for v-fps function and activation of the c-fps oncogenic potential are discussed.     

Autophosphorylation of soluble insulin receptor protein-tyrosine kinases. 1H NMR spectral changes observed during phosphorylation of mobile tyrosine residues.

Autophosphorylation of a soluble approximately 48-kDa derivative of the insulin receptor protein-tyrosine kinase occurs at multiple tyrosine residues (analogous to tyrosines 1158, 1162, and 1163 in the kinase homology region of the native receptor and tyrosines 1328 and 1334 in the carboxyl-terminal tail) and is accompanied by an increase in the specific activity of the enzyme toward exogenous substrates. A comparison of 1H NMR spectra of approximately 48- and approximately 38-kDa forms of enzyme (the latter generated by tryptic deletion of approximately 10 kDa from the carboxyl terminus of the approximately 48-kDa protein) allows a correlation of observed mobile tyrosine resonances to two of the known sites of autophosphorylation (residues 1328 and 1334). Furthermore, spectra acquired during autophosphorylation of the approximately 48-kDa enzyme reveal a rapid downfield shift in the resonances of these mobile tail tyrosines consistent with their phosphorylation (as confirmed by two-dimensional tryptic phosphopeptide mapping performed under identical conditions). This experimental strategy now provides a means by which to monitor protein-tyrosine kinase autophosphorylation in solution in real time.     

Deletion of approximately 10 kDa from the carboxyl terminus of a soluble approximately 48-kDa insulin receptor protein-tyrosine kinase results in slower rates of diphosphorylation of a series of dodecapeptide substrates. An assessment by 1H NMR.

Autophosphorylation of a soluble approximately 48-kDa derivative of the insulin receptor protein-tyrosine kinase is accompanied by an increase in its specific activity towards exogenous substrates. In the present study, we have utilized 1H NMR to compare the order and rate of mono- and diphosphorylation of multiple tyrosine residues in a series of synthetic dodecapeptide substrates (based on the receptor sequence, which includes major sites of autophosphorylation (RRDIYETDYYRK), with substitution(s) at positions 6 and/or 7 based on residue size and/or charge) by the approximately 48-kDa enzyme and by a approximately 38-kDa enzyme generated by tryptic deletion of approximately 10 kDa from the carboxyl terminus of the approximately 48-kDa protein. Both enzymes exhibit a marked order and progression of phosphorylation of peptide tyrosine residues; for each peptide, phosphorylation initiates and proceeds to completion first on tyrosine 9, followed by phosphorylation on tyrosine 10. Although removal of the carboxyl terminus does not affect the rate of monophosphorylation of these peptides on tyrosine 9, the smaller enzyme exhibits a slower rate of diphosphorylation (at tyrosine 10), as compared with the approximately 48-kDa enzyme.     

Activation of ZAP-70 through specific dephosphorylation at the inhibitory Tyr-292 by the low molecular weight phosphotyrosine phosphatase (LMPTP)

The ZAP-70 protein-tyrosine kinase plays a central role in signaling from the T cell antigen receptor. Recruitment and activation of ZAP-70 are transient and are terminated by phosphorylation of negative regulatory tyrosine residues and dephosphorylation of positively acting sites. We report that the low molecular weight protein-tyrosine phosphatase (LMPTP) specifically dephosphorylates the negative regulatory Tyr-292 of ZAP-70, thereby counteracting inactivation of ZAP-70. Expression of low levels of LMPTP resulted in increased ZAP-70 phosphorylation, presumably at the activating Tyr-493 and other sites, increased kinase activity, and augmented downstream signaling to the mitogen-activated protein kinase pathway. The ZAP-70 Y292F mutant was not affected by LMPTP. Our results indicate that LMPTP, like CD45, dephosphorylates a negative regulatory tyrosine site in a protein-tyrosine kinase and thereby strengthens T cell receptor signaling.