How do protein kinases recognize their substrates?
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Amino Acid Sequence↗
Biomedical subjects
L A Pinna
Publications and source records attributed to L A Pinna.
Phosphorylation of HIV-1 Rev protein: implication of protein kinase CK2 and pro-directed kinases.
HIV-1 Rev transactivator is readily phosphorylated at separate regions by protein kinase CK2 and MAP kinase. Protein kinase CK1 cannot replace CK2 as phosphorylating agent and cdc2 only slowly phosphorylates Rev at one of the two sites affected by MAP kinase. Mutational analysis shows that Ser-8 and, to a lesser extent, Ser-5 are phosphorylated by CK2. In contrast, a mutation (R14TV-->EED) which suppresses Rev activity dramatically enhances Rev phosphorylation either in vitro by CK2 or in vivo, suggesting that phosphorylation by CK2 could play a role in Rev down-regulation.
Amino Acid Sequence↗
The spleen protein-tyrosine kinase TPK-IIB is highly similar to the catalytic domain of p72syk.
TPK-IIB is a protein kinase that is predominant in the cytosol of spleen and is characterized by a high specific activity toward acidic peptide substrates and a low auto-phosphorylation activity. A prominent 52-kDa component purifies with the kinase [Marin, O., Donella-Deana, A., Brunati, A. M., Fischer, S. & Pinna, L. A. (1991) J. Biol. Chem. 266, 17798-17803]. Here we demonstrate that the 52-kDa protein displays sequence identity with the Miller-Dieker lissencephaly protein (LIS 1). The protein is not related to any known protein kinase and lacks an ATP-binding motif. The ATP binding and phosphotransferase activities of TPK-IIB can be fully accounted for by a minor 38-kDa protein band (p38/TPK-IIB) which can be separated from the 52-kDa protein by Mono-Q/FPLC in the presence of EDTA. Sequence analysis of p38/TPK-IIB reveals a high level of similarity, if not identity, with the catalytic domain of p72syk, a protein-tyrosine kinase implicated in the activation of hematopoietic cells. Antibodies raised against the catalytic domain of p72syk, but not antibodies raised against its N-terminal segment, cross-react with p38/TPK-IIB. The peptide substrate specificity of p72syk is almost identical to that of p38/TPK-IIB, which also supports the classification of TPK-IIB as a close relative (possibly a proteolytic or alternative spliced form) of p72syk. p38/TPK-IIB, however, exhibits a specific activity which is sixfold higher than that of p72syk and appears to be 50-fold more sensitive to inhibition by heparin. Thus, the observation that Ca(2+)-dependent degradation of p72syk by particulate fraction of rat spleen is accompanied by increased catalytic activity and increased sensitivity to heparin would be consistent with the possibility that hyperactive p38/TPK-IIB might be proteolytically generated from p72syk in response to an increase of intracellular Ca2+.
Affinity Labels↗
Protein kinase CK2 mutants defective in substrate recognition. Purification and kinetic analysis.
Five mutants of protein kinase CK2 alpha subunit in which altogether 14 basic residues were singly to quadruply replaced by alanines (K74A,K75A,K76A,K77A; K79A, R80A,K83A; R191A,R195A,K198A; R228A; and R278A, K279A,R280A) have been purified to near homogeneity either as such or after addition of the recombinant beta subunit. By this latter procedure five mutated tetrameric holoenzymes were obtained as judged from their subunit composition, sedimentation coefficient on sucrose gradient ultracentrifugation, and increased activity toward a specific peptide substrate as compared with the isolated alpha subunits. The kinetic constants and the phosphorylation efficiencies (V(max)/Km) of all the mutants with the parent peptide RRRADDSDDDDD and a series of derivatives, in which individual aspartic acids were replaced by alanines, have been determined. Three mutants, namely K74A,K75A,K76A,K77A; K79A, R80A,K83A; and R191A,R195A, K198A, display dramatically lower phosphorylation efficiency and 8-50-fold higher Km values with the parent peptide, symptomatic of reduced attitude to bind the peptide substrate as compared with CK2 wild type. Such differences either disappear or are attentuated if the mutants R191A,R195A, K198A; K79A,R80A,K83A; and K74A,K75A, K76A,K77A are assayed with the peptides RRRADDSADDDD, RRRADDSDDADD, and RRRADDSDDDAA, respectively. In contrast, the phosphorylation efficiencies of the other substituted peptides decrease more markedly with these mutants than with CK2 wild type. These data show that one or more of the basic residues clustered in the 191-198, 79-83, and 74-77 sequences are implicated in the recognition of the acidic determinants at positions +1, +3, and +4/+5, respectively, and that if these residues are mutated, the relevance of the other acidic residues surrounding serine is increased. In contrast the other two mutants, namely R228A and R278A,K279A, R280A, display with all the peptides V(max) values higher than CK2 wild type, counterbalanced however by somewhat higher Km values. It can be concluded from these data that all five mutations performed are compatible with the reconstitution of tetrameric holoenzyme, but all of them influence the enzymatic efficiency of CK2 to different extents. Although the basic residues mutated in the 74-77, 79-83, and 191-198 sequences are clearly implicated in substrate recognition by interacting with acidic determinants at variable positions downstream from serine, the other basic residues seem to play a more elusive and/or indirect role in catalysis.
Amino Acid Sequence↗
SH2 domains mediate the sequential phosphorylation of HS1 protein by p72syk and Src-related protein tyrosine kinases.
The protein tyrosine kinase p72syk readily phosphorylates hematopoietic linkage cell-specific protein p50/HS1 with high stoichiometry (up to 4 mol of Pi/mol of protein) and favorable kinetic constants (Km 77 nM, kcat 0.37 s-1), at sites that display the motif that is specifically recognized by the HS2 domains of Src tyrosine kinases. Such a phosphorylation converts p50/HS1 into a good substrate for c-Fgr, which in contrast is nearly inactive on nonphosphorylated p50/HS1. A phosphopeptide reproducing one of the main p50/HS1 site affected by p72syk, but neither its dephosphorylated derivative nor other phosphopeptides with different structure, blocks the secondary phosphorylation of phospho(p50/HS1) by c-Fgr but not its primary phosphorylation by p72syk. It also prevents the coimmunoprecipitation of phospho(HS1) with c-Fgr by anti-(c-Fgr) antibodies. In contrast the HS1[393-402] phosphopeptide is ineffective on the kinase activity of c-Fgr when tested with peptide substrates, showing that inhibition of p50/HS1 phosphorylation is not exerted at the catalytic site of c-Fgr. The sequential phosphorylation of p50/HS1 as well as its specific blockage by the HS1 phosphopeptide is also observable if c-Fgr is replaced by two other Src-related kinases, namely, Lyn and Fyn, as secondary phosphorylating agents. None of these Src-related kinases, however, can carry out the phosphorylation of p50/HS1 at the sites affected by p72syk, even after prolonged incubation. Our data suggest that sequential phosphorylation might represent a general mechanism by which p72syk and other Syk-related kinases generate substrates for Src-related protein tyrosine kinases. They also show that sequential phosphorylation (requiring the concerted action of a primary and a secondary kinases) cannot be surrogated by "processive" phosphorylation where a single kinase catalyzes both the primary and secondary phosphorylation, although both these modes of multiple phosphorylation are based on interactions between SH2 domains of the kinases and phosphotyrosyl sites of the substrate.
Adaptor Proteins, Signal Transducing↗
Golgi apparatus mammary gland casein kinase: monitoring by a specific peptide substrate and definition of specificity determinants.
The casein kinase from the Golgi apparatus of lactating mammary gland (GEF-CK) is distinct from ubiquitous 'casein kinases' termed protein kinases CK1 and CK2 and appears to define a family of secretory pathways protein kinases that phosphorylate seryl residues followed by an acidic residue at position +2. In this report we show that a new synthetic peptide substrate derived from beta-casein (beta[28-40]) is suitable for the fast, efficient and selective monitoring of GEF-CK, being unaffected by CK1 and CK2, and we define the consensus sequence of this protein kinase as being Ser-Xaa-Glu/SerP, distinct from that of CK2 (Ser/Thr-X-X-Glu/Asp/SerP/TyrP). In particular, the failure to recognize Asp as crucial specificity determinant prevents the phosphorylation of the specific CK2 peptide substrate RRRADDSDDDDD by GEF-CK. Thus, peptide substrates are now available for the fast and specific monitoring of all the three classes of 'casein kinases', CK1, CK2 and GEF-CK.
Amino Acid Sequence↗
A comparative study of the phosphotyrosyl phosphatase specificity of protein phosphatase type 2A and phosphotyrosyl phosphatase type 1B using phosphopeptides and the phosphoproteins p50/HS1, c-Fgr and Lyn.
The phosphotyrosyl phosphatase (PTPase) specificity of phosphotyrosyl-phosphatase-activator-(PTPA)-stimulated protein phosphatase (PP)2A(D) (rabbit muscle) and a bona fide PTP-1B (Xenopus laevis oocytes) were examined in vitro using phosphotyrosine-containing peptides, derived from the phosphorylation sites of p34cdc2, p50/HS1 protein, Abl, c-Src and c-Fgr, as well as the intact phosphoprotein p50/HS1 and the Src-related tyrosine kinases, Lyn and c-Fgr. The local specificity determinants were found to be different for both PTPases. The length of the phosphopeptides is more important for PP2A(D) than for PTP-1B, C-terminal acidic residues adjacent to the phosphotyrosine are detrimental for the PTPase activity of PP2A(D), but they do not affect the PTP-1B activity. Acidic residues at the --2 and --3 position relative to Tyr(P) primarily dictate dephosphorylation by PTP-1B. The higher-order structure of the protein substrates also differentially influences both enzymes: the phospho-octapeptide KDDEYpNPA, which reproduces the autophosphorylation site in c-Fgr (Tyr400), is only dephosphorylated by PP2A(D) if embedded in the intact protein, whereas the opposite is true for PTP-1B. Both the intact p50/HS1 phosphoprotein and the derived phosphopeptide are substrates only for PTP-1B and not for PP2A(D). Lyn and c-Fgr phosphorylated by C-terminal Src kinase (CSK) at their down-regulatory site are resistant to the action of both PTPases while the [Phe6]Src-(514-533) phosphopeptide, representing the highly similar site affected by CSK in c-Src, is readily dephosphorylated by both PTPases, although to a different extent. In vitro dephosphorylation of the c-Fgr Tyr400 site by PP2A(D) is correlated with a decreased tyrosine kinase activity towards exogenous substrates. Under experimental conditions in which both Tyr400 (autophosphorylation site) and Tyr511 (down-regulatory site) of c-Fgr are phosphorylated, PP2A(D) can reverse both phosphorylations.
Amino Acid Sequence↗
Mapping the residues of protein kinase CK2 alpha subunit responsible for responsiveness to polyanionic inhibitors.
The quadruple mutation of the whole basic cluster, K74KKK77 conserved in the catalytic subunits of protein kinase CK2 and implicated in substrate recognition, not only abolishes inhibition by heparin but even induces with some peptide substrates an up to 5-fold stimulation by heparin in the 0.5-5 micrograms/ml concentration range. Two other mutants defective in substrate recognition, R191, 195K198A and K79R80K83A, display either a 100-fold reduction or no alteration at all in heparin inhibition, respectively. In contrast sensitivity to heparin inhibition is increased 30-fold by a single mutation affecting Arg-228 while it is not altered by a triple mutation in the small insert of subdomain XI (mutant R278K279R280A). The effect of the same mutations on inhibition by pseudosubstrate EEEEEYEEEEEEE is different, the mutant displaying the most reduced sensitivity being R191,195K198A, followed by K74-77A and K79R80K83A; the other mutants are almost indistinguishable from CK2 wild type. Substantial reduction of inhibition by poly(Glu,Tyr)4:1 is only observable with mutant R191,195K198A, whereas R228A is significantly more sensitive to inhibition. These data show that the mode of inhibition of CK2 by polyanionic compounds occurs through substantially different mechanisms involving residues that are variably concerned with substrate recognition.
Amino Acid Sequence↗
A membrane-bound protein kinase from rabbit reticulocytes is an active form of multipotential S6 kinase.
An active ribosomal protein S6 kinase has been highly purified from the membranes of rabbit reticulocytes by chromatography of the Triton X-100 extract on DEAE-cellulose, SP-Sepharose Fast Flow, and by FPLC on Mono Q and Superose-12. The S6 kinase elutes around 40 000 daltons upon gel filtration on Superose-12 or Sephacryl S-200. It has a subunit molecular weight of 40-43 kDa as determined by protein kinase activity following denaturation/renaturation in SDS-polyacrylamide gels containing S6 peptide. It also phosphorylates translational initiation factors eIF-2 and eIF-4F, glycogen synthase, histone 1, histone 2B, myelin basic protein, but not prolactin, skeletal myosin light chain, histone 4, tubulin, and casein. Apparent Km values have been determined to be 15 microM for ATP, 1.2 microM for S6 and 10 microM for S6 peptide. Two-dimensional tryptic phosphopeptide mapping shows the same sites on S6 are phosphorylated as those identified previously with proteolytically activated multipotential S6 kinase from rabbit reticulocytes, previously denoted as protease activated kinase II. Examination of relative rates of phosphorylation and kinetic constants of synthetic peptides based on previously identified phosphorylation sites, indicates a minimum substrate recognition sequence to be arginine at the n - 3 position. Based on these characteristics, including molecular weight and an expanded substrate specificity, the membrane S6 kinase can be distinguished from the p90 (Type I) and p70 (Type II) S6 kinases, and from protein kinase C and the catalytic subunit of cAMP-dependent protein kinase.
Amino Acid Sequence↗
Isolation from spleen of a 57-kDa protein substrate of the tyrosine kinase Lyn. Identification as a protein related to protein disulfide-isomerase and localisation of the phosphorylation sites.
A 57-kDa protein (p57) has been purified to homogeneity from a microsomal fraction of rat spleen. It is specifically and efficiently phosphorylated by the Src-like tyrosine kinase Lyn purified from the same source with a Km of 0.34 microM. The tyrosine kinases c-Fgr, Fyn, C-terminal Src kinase and p72syk, as well as the Ser/Thr-specific cAMP-dependent protein kinase and protein kinases CK1 and CK2 do not phosphorylate p57. C-terminal Src kinase, which acts to down-regulate the Src-like protein-tyrosine kinases, almost completely prevents the protein phosphorylation catalysed by Lyn. Protein mass fingerprinting with tryptic fragments identified p57 as a protein related to protein disulfide-isomerase which belongs to the superfamily of Cys-Gly-His-Cys-containing sequences. Lyn phosphorylates tyrosine residues Y444, Y453 and Y466 which are located in a highly acidic region of the protein at the C-terminus. Upon phosphorylation, p57 forms a complex with Lyn which can be immunoprecipitated with anti-Lyn IgG. The association which occurs between the phosphorylated substrate and the SH2 domain of the kinase is consistent with the suggested 'processive phosphorylation' model, which implies that a primary phosphorylation site of the substrate binds to the SH2 domain of the enzyme and triggers the phosphorylation at secondary site(s).
Amino Acid Sequence↗
Solid phase synthesis of pp60src-related phosphopeptides via 'global' phosphorylation and their use as substrates for enzymatic phosphorylation by casein kinase-2.
The seven phosphopeptide derivatives based on the native -NEYTA- sequence of the pp60src protein kinase family, Asn-Glu-Tyr(P)-Ser-Ala, Ala-Glu-Tyr(P)-Ser-Ala, Ala-Ser-Tyr(P)-Ser-Ala, Ala-Ser(P)-Tyr-Ser-Ala, Ala-Thr-Tyr(P)-Ser-Ala, Ala-Thr(P)-Tyr-Ser-Ala and Ala-Ser(P)-Tyr(P)-Ser-Ala, were prepared in good yield using the "global' "phosphite-triester' phosphorylation method. The peptide resins were assembled using the Fmoc mode of solid phase peptide synthesis (PyBOP coupling method) with specific Ser-, Thr-, or Tyr-residues incorporated as their side chain free Fmoc-derivatives. The final "global' phosphorylation of the peptide resins was accomplished using di-tert-butyl N, N-diethylphosphoramidite followed by m-chloroperoxybenzoic acid oxidation of the resultant di-t-butyl phosphite triester intermediate. Subsequent resin cleavage and deprotection of the phosphorylated peptide resins was effected by treatment with 5% anisole: TFA and gave the seven phosphopeptides in high yield and purity. The use of the seven synthetic phosphopeptides in enzymatic (casein kinase-2) phosphorylation studies showed that, (A) the change of the target Thr site to Ser resulted in markedly improved phosphorylation of the peptide substrates, (B) that the Tyr(P) residue in the - 1 position was significantly more important than the Ser(P)/Thr(P) residue in the - 2 position for efficient seryl phosphorylation, and (C) that an acidic residue in the - 2 position relative to the target site facilitated phosphorylation of the downstream seryl residue irrespective of the nature of the acidic residue in the -Xxx-Tyr(P)-Ser- and -Xxx-Tyr-Ser- sequences {Xxx = Ser(P), Thr(P), Glu}. In addition to the Tyr(P) residue directing phosphorylation to the +1 position, the good phosphorylation of both ASY(P)SA and ATY(P)SA by casein kinase-2 indicated that the Tyr(P) residue was also able to direct phosphorylation to a Ser/Thr in the - 1 position.
Amino Acid Sequence↗
Phosphotyrosine specifies the phosphorylation by protein kinase CK2 of a peptide reproducing the activation loop of the insulin receptor protein tyrosine kinase.
Protein kinase CK2 is a ubiquitous Ser/Thr-specific protein kinase responsible for the phosphorylation of many proteins implicated in signal transduction. It phosphorylates both threonyl and seryl residue(s) of the insulin receptor beta-subunit. In this study, a series of peptides, reproducing all the threonyl sites of the intracellular domain of the insulin receptor that display the consensus sequence for CK2, has been synthesized and used as substrate for purified rat liver CK2. The only peptide readily phosphorylated is the one reproducing the activation loop of the insulin receptor (EIYET1160DYYA), including three tyrosines (Y1158, Y1162 and Y1163) whose phosphorylation through an intermolecular autocatalytic process promotes the activation of the receptor kinase. The phosphorylation efficiency of T1160 is increased almost 20-fold if these three tyrosines are previously phosphorylated. By using variably phosphorylated peptides, the tyrosine mainly responsible for such a hierarchical phosphorylation process has been identified as Y1163. It can be concluded, from these data, that T1160 situated in the activation loop of the insulin receptor, represents an excellent target for CK2, its phosphorylation being triggered by the previous autophosphorylation of the three tyrosyl residues surrounding it, with special reference to Y1163. These data are consistent with the implication of CK2 in the regulation of the activation process of the insulin receptor protein tyrosine kinase.
Animals↗
Site specificity of p72syk protein tyrosine kinase: efficient phosphorylation of motifs recognized by Src homology 2 domains of the Src family.
Protein tyrosine kinase p72syk purified from rat spleen has been assayed for its ability to phosphorylate a number of peptide substrates derived from naturally occurring phospho-acceptor sites. The phosphorylation efficiency is extremely variable, depending on the peptide sequence, with Km values in the 3-1500 microM range. The by far best peptide substrates, with Km values of 3 and 4 microM are those reproducing the phospho-acceptor sites of Vav and HS1 proteins, respectively. These sites include multiple acidic residues flanking tyrosine on both sides and they also display the consensus sequences (YEDL and YEEV) preferred by the SH2 domains of the Src family. Alteration of this consensus in the HS1 peptide, by replacing either the glutamic acid or valine, also reduces the phosphorylation efficiency by p72syk. Also the replacement of acidic residues at position -1 and, to a lesser extent at positions -3 and -4 (but not at positions +3 and +5) are detrimental. These observations may suggest a role of p72syk in the recruitment of ligands/substrates for the Src family enzymes. We also show that the HS1 peptide can be used for the specific monitoring of p72syk since neither the two Src-related c-Fgr and Lyn kinases (needing a hydrophobic instead of acidic residue at position -1) nor CSK appreciably phosphorylate it.
Adaptor Proteins, Signal Transducing↗
The Ste locus, a component of the parasitic cry-Ste system of Drosophila melanogaster, encodes a protein that forms crystals in primary spermatocytes and mimics properties of the beta subunit of casein kinase 2.
Males of Drosophila melanogaster lacking the Y chromosome-linked crystal locus show multiple meiotic alterations including chromosome disorganization and prominent crystal formation in primary spermatocytes. These alterations are due to the derepression of the X chromosome-linked Stellate sequences. To understand how the derepression of the Stellate elements gives rise to these abnormalities, we have expressed the protein encoded by the Stellate sequences in bacteria and produced an antibody against the fusion protein. Immunostaining of crystal- testes has clearly shown that the Stellate protein is a major component of the crystals. Moreover, in vitro experiments have shown that this protein can interact with the catalytic alpha subunit of casein kinase 2 enzyme, altering its activity.
Amino Acid Sequence↗
Phosphorylation and activation of protein kinase CK2 by p34cdc2 are independent events.
Recombinant isolated beta-subunit of protein kinase CK2 is readily phosphorylated by p34cdc2/cyclin B kinase at Ser209 with favourable kinetic constants (Km = 1.7 microM, Vmax = 20 nmol.min-1.mg-1). Two synthetic peptides reproducing the 170-215 and the 206-215 C-terminal fragments of the beta-subunit are also phosphorylated though with tenfold higher Km values (19.5 and 28.0 microM, respectively). In contrast, both the beta-subunit associated with the alpha-subunit to give the heterotetrameric holoenzyme and the native CK2 are not appreciably phosphorylated by p34cdc2. These data suggest that the Ser209 beta-subunit phosphorylation observed in intact cells occurs prior to beta-subunit incorporation into the holoenzyme. The isolated CK2 alpha-subunit is not phosphorylated to any appreciable extent by p34cdc2 kinase. Its catalytic activity is nevertheless increased up to fivefold upon incubation with p34cdc2/cyclin B kinase complex. Such a stimulation of activity is comparable to that induced by the beta-subunit and it is paralleled by a 40% decrease of p34cdc2/cyclin B catalytic activity. Similar to beta-subunit, p34cdc2/cyclin B also protects the alpha-subunit against thermal inactivation. CK2 holoenzyme is also stimulated by p34cdc2/cyclin B, albeit less dramatically than the isolated alpha-subunit. Such an effect is also evident with CK2 holoenzyme reconstituted with a mutated beta-subunit lacking the p34cdc2 phosphorylation site and it is not accompanied by any appreciable phosphorylation of either the beta or the alpha-subunit. These data indicate that in vitro CK2 alpha-subunit interacts with and is activated by p34cdc2/cyclin B kinase by a mechanism that does not imply the phosphorylation of CK2.
Amino Acid Sequence↗
Photosensitized inhibition of growth factor-regulated protein kinases by hypericin.
The naphthodianthrone hypericin causes a photosensitized inhibition of protein kinases involved in growth factor signalling pathways. Nanomolar concentrations of hypericin inhibit the protein tyrosine kinase activities (PTK) of the epidermal growth factor receptor and the insulin receptor, while being ineffective towards the cytosolic protein tyrosine kinases Lyn, Fgr, TPK-IIB and CSK. Photosensitized inhibition by hypericin is not restricted to receptor-PTKs since the Ser/Thr protein kinases (protein kinase CK-2, protein kinase C and mitogen-activated kinase) are also extremely sensitive to inhibition (IC50 value for protein kinase CK-2 = 6 nM). A comparison of the hypericin-mediated inhibition of the epidermal growth factor-receptor PTK and protein kinase CK-2 revealed that the inhibition is irreversible, strictly dependent upon irradiation of the enzyme-inhibitor complex with fluorescent light and likely mediated by the formation of radical intermediates (type I mechanism). Although the exact molecular basis for the selectivity of enzyme inhibition by hypericin remains unknown, our results suggest that distantly related protein kinases could still share common reactive domains for the interaction with hypericin.
Amino Acid Sequence↗
Dissection of the dual function of the beta-subunit of protein kinase CK2 ('casein kinase-2'): a synthetic peptide reproducing the carboxyl-terminal domain mimicks the positive but not the negative effects of the whole protein.
The dual function of the regulatory beta-subunit of protein kinase CK2 is highlighted by its ability to abolish calmodulin phosphorylation in contrast to its stimulatory effect on the phosphorylation of peptide substrates. Here we show that a synthetic peptide reproducing the C-terminal region of the beta-subunit (beta[170-215]) stimulates to a similar extent the phosphorylation of either the peptide substrate or calmodulin and also protects the catalytic alpha-subunit against thermal inactivation as efficiently as full-length beta-subunit. These data show that the positive and negative functions of the beta-subunit reside in physically separated domains and that the elements responsible for positive regulation are located in the C-terminal region.
Amino Acid Sequence↗
Hierarchical phosphorylation of a 50-kDa protein by protein tyrosine kinases TPK-IIB and C-Fgr, and its identification as HS1 hematopoietic-lineage cell-specific protein.
TPK-IIB is an acidophilic protein tyrosine kinase devoid of autophosphorylation activity and unrelated to the Src family kinases [Marin, O., Donella-Deana, A., Brunati, A.M., Fischer, S. & Pinna, L. A. (1991) J. Biol. Chem. 266, 17798-17803]. Here, we describe the purification to homogeneity of a 50-kDa prominent substrate (p50) of TPK-IIB from rat spleen homogenates. Microsequence analysis of fragments from purified p50 discloses its homology to HS1, a hematopoietic-lineage cell-specific protein implicated in B-cell-antigen receptor-mediated signalling [Yamanashi, Y., Okada, M., Semba, T., Yamori, T., Umemori, H., Tsunasawa, S., Toyoshima, K., Kitamura, D., Watanabe, T. & Yamamoto, T. (1993) Proc. Natl Acad. Sci. USA 90, 3631-3635]. p50 is an excellent substrate for TPK-IIB, exhibiting very favourable kinetic constants (Km = 0.07 microM, kcat = 1.5) and incorporating up to 4 mol P/mol protein. p50 is, however, a weak substrate for the Src-related protein kinases Lyn and c-Fgr. Once phosphorylated by TPK-IIB, however, p50 is converted into a good substrate for c-Fgr and, to a lesser extent, for Lyn. p50 phosphorylated by TPK-IIB associates with c-Fgr and Lyn, as judged by co-immunoprecipitation with anti-Fgr IgG and anti-Lyn IgG, respectively. These data suggest the involvement of TPK-IIB in B-cell-antigen receptor-mediated signalling, and support the idea that phosphorylation by TPK-IIB might be a prerequisite for the recruitment of certain protein substrates by Src-related protein tyrosine kinases.
Adaptor Proteins, Signal Transducing↗