Biomedical subjects

O Marin

Publications and source records attributed to O Marin.

At least 37 records · Page 2Linked to original sources

PubMed1999

Myotonic dystrophy protein kinase expressed in rat cardiac muscle is associated with sarcoplasmic reticulum and gap junctions.

Myotonic dystrophy (DM) is one of the most prevalent muscular diseases in adults. The molecular basis of this autosomal disorder has been identified as the expansion of a CTG repeat in the 3' untranslated region of a gene encoding a protein kinase (DMPK). The pathophysiology of the disease and the role of DMPK are still obscure. It has been previously demonstrated that DMPK is localized at neuromuscular junctions, myotendinous junctions, and terminal cisternae of the sarcoplasmic reticulum (SR), in the skeletal muscle, and at intercalated discs in the cardiac muscle. We report here new findings about specific localization of DMPK in the heart. Polyclonal antibodies raised against a peptide sequence of the human DMPK were used to analyze the subcellular distribution of the protein in rat papillary muscles. Confocal laser microscopy revealed a strong although discontinuous reactivity at intercalated discs, together with transverse banding on the sarcoplasm. At higher resolution with immunogold electron microscopy, we observed that DMPK is localized at the cytoplasmic surface of junctional and extended junctional sarcoplasmic reticulum, suggesting that DMPK is involved in the regulation of excitation-contraction coupling. Along the intercalated disc, DMPK was found associated with gap junctions, whereas it was absent in the two other kinds of junctional complexes (fasciae adherentes and desmosomes). Immunogold labeling of gap junction purified fractions showed that DMPK co-localized with connexin 43, the major component of this type of intercellular junctions, suggesting that DMPK plays a regulatory role in the transmission of signals between myocytes.

Animals↗

PubMed1999

Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum.

The telencephalon is organized into distinct longitudinal domains: the cerebral cortex and the basal ganglia. The basal ganglia primarily consists of a dorsal region (striatum) and a ventral region (pallidum). Within the telencephalon, the anlage of the pallidum expresses the Nkx2.1 homeobox gene. A mouse deficient in Nkx2.1 function does not form pallidal structures, lacks basal forebrain TrkA-positive neurons (probable cholinergic neurons) and has reduced numbers of cortical cells expressing GABA, DLX2 and calbindin that migrate from the pallidum through the striatum and into the cortex. We present evidence that these phenotypes result from a ventral-to-dorsal transformation of the pallidal primordium into a striatal-like anlage.

Animals↗

PubMed1999

A multifunctional network of basic residues confers unique properties to protein kinase CK2.

Protein kinase CK2 is characterized by a number of features, including substrate specificity, inhibition by polyanionic compounds and intrasteric down-regulation by its beta-subunit, which denote a special aptitude to interact with negatively charged ligands. This situation may reflect the presence in CK2 catalytic subunits of several basic residues that are not conserved in the majority of other protein kinases. Some of these residues, notably K49 in the 'Gly rich loop', K74, K75, K76, K77, K79, R80, K83 in the 'Lys rich segment' and R191, R195, K198 in the 'p+1 loop', have been shown by mutational studies to be implicated to various extents and with distinct roles in substrate recognition, inhibition by heparin and by pseudosubstrate and instrasteric regulation. Molecular modelization based on crystallographic data provide a rationale for the biochemical observations, showing that several of these basic residues are clustered around the active site where they make contact with individual acidic residues of the peptide substrate. They can also mediate the effect of polyanionic inhibitors (e.g. heparin) and of regulatory elements present in the beta-subunit, in the N terminal segment of the catalytic subunit and possibly in other proteins interacting with CK2. Our data also disclose a unique mode of binding of the phosphoacceptor substrate which bridges across the catalytic cleft making contacts with both the lower and upper lobes of CK2.

Amino Acid Sequence↗

PubMed1999

Functional analysis of CK2beta-derived synthetic fragments.

Synthetic peptides reproducing the amino and carboxyl terminal region of CK2beta subunit have been analyzed for their ability to mimic different properties of full length beta subunit. Peptide beta[1-77], containing both the autophosphorylation site and the down-regulatory domain 55-64, is readily phosphorylated by alpha subunit whose activity is concomitantly inhibited. Such inhibition is accompanied by a weak interaction detectable by BIAcore sensograms but not by far Western blots, and is not reversed by polylysine which conversely overcome inhibition of calmodulin phosphorylation by full length beta subunit. A strong interaction with alpha is observed with beta[155-215] but not with its shorter derivative beta[170-215] as judged from far Western blotting and sucrose gradient ultracentrifugation analysis. Both peptides, however, affect the regular interaction between alpha and beta subunits altering the autophosphorylation pattern and responsiveness to salt. beta[155-215], unlike beta[170-215] tends to aggregate more readily than full length beta subunit. This behaviour which is reminiscent of the homodimerization of full length beta subunit, would indicate that tight self-association of beta[155-215] crucially depends on residues in the 155-170 sequence. Failure of beta[1-77] fragment to mediate responsiveness to polybasic peptides and accentuated self-association propensity of beta[155-215] suggest that other structural elements between the sequences 1-77 and 155-215 are required in order to confer optimal functionality to the beta subunit.

Amino Acid Sequence↗

PubMed1998

Biochemical evidence that the N-terminal segments of the alpha subunit and the beta subunit play interchangeable roles in the activation of protein kinase CK2.

The concept that the amino-terminal segment plays a role in conferring high basal activity to protein kinase CK2 alpha subunit has been validated by generating two mutants (Y26F and delta2-6) which are defective both in catalytic activity and in thermal stability. The additional finding that the activity of the two mutants is fully restored upon association with the regulatory beta subunit, in conjunction with the observation that synthetic peptides reproducing the N-terminal segment (1-30) and the activation loop (175-201) of CK2alpha counteract the functional effects of the C-terminal domain of the beta subunit, is consistent with a mechanism of activation of CK2 where the N-terminal domain of alpha and the C-terminal domain of beta play interchangeable roles.

Amino Acid Sequence↗

PubMed1998

Spontaneous autophosphorylation of Lyn tyrosine kinase at both its activation segment and C-terminal tail confers altered substrate specificity.

Two tyrosyl residues have been reported to play a crucial role in the regulation of protein tyrosine kinases of the Src family: autophosphorylation of Tyr416 (c-Src numbering) located in the catalytic domain correlates with enzyme activation, while Csk-mediated phosphorylation of the C-terminal tyrosine Tyr527 (c-Src numbering) gives rise to inactive forms of Src kinases. Here we show that the Src-related Lyn kinase undergoes spontaneous and stoichiometric autophosphorylation at both Tyr396 (homologous to c-Src Tyr416) and Tyr507 (homologous to c-Src Tyr527). Such a doubly autophosphorylated form of Lyn is hyperactive toward peptide substrates and insensitive to Csk-induced downregulation. In contrast, doubly autophosphorylated Lyn exhibits reduced activity toward protein substrates such as phospho-p50/HS1 (hematopoietic-lineage cell-specific protein) and p57/PDI (protein disulfide isomerase related protein), whose multiple sequential/processive phosphorylation relies on the accessibility of the SH2 domain of the kinase. These data disclose a novel conformation of Lyn that is catalytically active despite the presence of an intramolecular interaction between the phosphorylated tail and the SH2 domain. This enzyme conformation is expected to display a reduced oncogenic potential resulting from its defective recognition of a subset of protein substrates whose targeting is mediated by the Lyn SH2 domain.

Amino Acid Sequence↗

PubMed1998

Chemical synthesis and structural characterization of the RGD-protein decorsin: a potent inhibitor of platelet aggregation.

Decorsin is a 39-residue RGD-protein crosslinked by three disulfide bridges isolated from the leech Macrobdella decora belonging to the family of GPIIb-IIIa antagonists and acting as a potent inhibitor of platelet aggregation. Here we report the solid-phase synthesis of decorsin using the Fmoc strategy. The crude polypeptide was purified by reverse-phase HPLC in its reduced form and allowed to refold in the presence of glutathione. The homogeneity of the synthetic oxidized decorsin was established by reverse-phase HPLC and capillary zone electrophoresis. The results of amino acid analysis after acid hydrolysis of the synthetic protein, NH2-terminal sequencing and mass determination (4,377 Da) by electrospray mass spectrometry were in full agreement with this theory. The correct pairing of the three disulfide bridges in synthetic decorsin was determined by a combined approach of both peptide mapping using proteolytic enzymes and analysis of the disulfide chirality by CD spectroscopy in the near-UV region. Synthetic decorsin inhibited human platelet aggregation with an IC50 of approximately 0.1 microM, a figure quite similar to that determined utilizing decorsin from natural source. In particular, the synthetic protein was 2,000-fold more potent than a model RGD-peptide (e.g., Arg-Gly-Asp-Ser) in inhibiting platelet aggregation. Thermal denaturation experiments of synthetic decorsin, monitored by CD spectroscopy, revealed its high thermal stability (Tm approximately 74 degrees C). The features of the oxidative refolding process of reduced decorsin, as well as the thermal stability of the oxidized species, were compared with those previously determined for the NH2-terminal core domain fragment 1-41 or 1-43 from hirudin. This fragment shows similarity in size, pairing of the three disulfides and three-dimensional structure with those of decorsin, even if very low sequence similarity. It is suggested that the less efficient oxidative folding and the enhanced thermal stability of decorsin in respect to those of hirudin core domain likely can be ascribed to the presence of the six Pro residues in the decorsin chain, whereas none is present in the hirudin domain. The results of this study indicate that decorsin can be obtained by solid-phase methodology in purity and quantities suitable for structural and functional studies and thus open the way to prepare by chemical methods novel decorsin derivatives containing unusual amino acids or even non-peptidic moieties.

Amino Acid Sequence↗

PubMed1997

Mutational analysis of residues implicated in the interaction between protein kinase CK2 and peptide substrates.

Sixteen derivatives of the optimal peptide substrate RRRA-DDSDDDDD in which aspartic acids were singly or multiply substituted by alanine have been assayed for their phosphorylation efficiency by either wild type protein kinase CK2 or CK2 alpha mutants defective in substrate recognition. With wild type CK2, the only detrimental single substitutions were those at positions +3 and +1. Each of these caused a 5-fold increase of Km and a 2-fold decrease of the Vmax values. If both aspartic acids at n + 1 and n + 3 were substituted however, the Km rose 24-fold and the Vmax decreased 16-fold. Multiple substitutions tend to have a more than additive effect even if they affect individually dispensable aspartic acids; thus, double, triple, and quintuple substitutions at positions n - 2 and -1, and n + 2, +4, and +5 had detrimental consequences comparable to those observed with substitutions at n + 1 and n + 3. These data indicate that additional acidic residues besides those at n + 1 and n + 3 are collectively required for efficient phosphorylation of CK2 substrates. They are also consistent with a flexible mode of binding of the substrate, where acidic residues may play interchangeable roles. Among twelve CK2 mutants in which basic residues suspected to be implicated in substrate recognition have been replaced by alanine, only K74-77A, K79R80K83A, R191,195K198A, and K198A showed substantially increased Km values with the optimal substrate RRRA-DDSDDDDD, symptomatic of a reduced ability to bind it. However, if the suboptimal substrate RRRA-AASDDDDD was used, the single mutants K49A, K71A, K77A, R80A, and H160A also exhibited Km values significantly higher than those of wild type CK2. Kinetic analysis with singly substituted derivatives of peptide RRRA-DDSDDDDD revealed that K49 is implicated in the recognition of the determinant at position n + 2, K77 cooperates with other residues nearby in the interaction with the determinants at n + 3 and n + 4, while K198 plays a prominent role in the recognition of the determinant at n + 1.

Binding Sites↗

PubMed1997

Biochemical evidence that Saccharomyces cerevisiae YGR262c gene, required for normal growth, encodes a novel Ser/Thr-specific protein kinase.

Saccharomyces cerevisiae YGR262c gene, whose disruption causes severely defective growth, encodes a putative protein kinase shorter than any other protein kinase biochemically characterized to date and lacking some of the conserved features of these enzymes. Here we show that the product of the YGR262c gene, piD261, expressed in E. coli with a C-terminal (His)6 tag, is a bona fide Ser/Thr protein kinase as judged from its capability to autophosphorylate and to phosphorylate casein and osteopontin in the presence of [gamma-32P]ATP. In contrast, no phosphorylation of histones, myelin basic protein, phosvitin, bovine serum albumin and poly(Glu/Tyr)4:1 could be detected. Mn2+ or, less effectively, Co2+ are required for piD261 catalytic activity, which is conversely undetectable in the presence of Mg2+, a behaviour unique among Ser/Thr protein kinases.

Amino Acid Sequence↗

PubMed1997

Basic residues in the 74-83 and 191-198 segments of protein kinase CK2 catalytic subunit are implicated in negative but not in positive regulation by the beta-subunit.

Protein kinase CK2 is a ubiquitous pleiotropic serine/threonine protein kinase whose holoenzyme is comprised of two catalytic (alpha and/or alpha') and two non-catalytic, beta-subunits. The beta-subunit possesses antagonist functions that can be physically dissected by generating synthetic fragments encompassing its N-terminal and C-terminal domains. Here we show that by mutating basic residues in the 74-77 and in the 191-198 regions of the alpha-subunit, the negative regulation by the beta-subunit and by its N-terminal synthetic fragment CK2beta-(1-77), which is observable using calmodulin as a substrate for phosphorylation, is drastically reduced. In contrast, the positive regulation by a C-terminal, CK2beta-(155-215)-peptide is unaffected or even increased. Moreover, the basal activity of alpha mutants K74-77A, K79R80K83A, and R191R195K198A toward specific peptide substrates is stimulated by the beta-subunit many fold more than that of alpha wild type, while extrastimulation by beta mutant D55L56E57A, observable with alpha wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of beta is mediated by basic residues in the 74-83 and in the 191-198 sequences of the alpha-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the beta subunit operates as a pseudosubstrate. In contrast, another CK2alpha mutant, V66A, is more sensitive to inhibition by either beta-subunit or its N-terminal, CK2beta-(1-77)-peptide, while its stimulation by the C-terminal peptide, CK2beta-(155-215), is comparable to that of alpha wild type. These observations suggest an indirect role of Val66 in conferring to the alpha-subunit a conformation less sensitive to down regulation by beta-subunit.

Amino Acid Sequence↗

PubMed1997

Physical dissection of the structural elements responsible for regulatory properties and intersubunit interactions of protein kinase CK2 beta-subunit.

The noncatalytic beta-subunit of protein kinase CK2 has been shown to display various and in some respects antagonistic effects on the catalytic alpha-subunit [Boldyreff et al. (1993) Biochemistry 32, 12672-12677; Meggio et al. (1994) Biochemistry 33, 4336-4342]. We have now examined the ability of peptides encompassing the N- and C-terminal regions of the beta-subunit (beta[1-77] and beta[155-215]) to mimic the functions of the whole-length beta-subunit. Peptide beta[155-215] possesses only the positive features of the beta-subunit in that it prevents thermal inactivation and stimulates basal activity of the alpha-subunit, while it does not inhibit but rather stimulates calmodulin phosphorylation. In sharp contrast, peptide beta[1-77] neither protects the alpha-subunit nor stimulates its basal activity, while acting as a powerful and specific inhibitor of calmodulin phosphorylation. Peptide beta[155-215], but not peptide beta[1-77], stably interacts with alpha-subunit and also displays remarkable self-associating properties. A shorter derivative of beta[155-215], beta[170-215], displaying weaker stimulatory properties fails to stably interact with the alpha-subunit and to give rise to dimeric/multimeric forms. These data show that the elements responsible for the negative regulation are concentrated in the N-terminal moiety of the beta-subunit, whereas the C-terminal region retains the beneficial properties of the beta-subunit and is capable of self-association and binding of the alpha-subunit. Residues between 155 and 170 are necessary for the latter functions.

Amino Acid Sequence↗

PubMed1997

Sequence specificity of C-terminal Src kinase (CSK)--a comparison with Src-related kinases c-Fgr and Lyn.

An eicosapeptide encompassing the C-terminal tail of c-Src (Tyr527) which is conserved in most Src-related 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 C-terminal segments of c-Src mutants defective in CSK phosphorylation [MacAuley, A., Okada, M., Nada, S., Nakagawa, H. & Cooper, J. A. (1993) Oncogene 8, 117-124] AFLEDSCTGTEPLYQRGENL (mutant number 28) and AFLEDNFTGTKPQYHPGENL (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(m) 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(m) value (63 microM). 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, TPKIIB/p38syk, related to the catalytic domain of p72syk, selects acidic residues at nearly all positions, n-1 included. These data support the notion that the features determining the specific phosphorylation of the C-terminal 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.

Amino Acid Sequence↗

PubMed1997

Specific stimulation of c-Fgr kinase by tyrosine-phosphorylated (poly)peptides--possible implication in the sequential mode of protein phosphorylation.

Hematopoietic lineage cell-specific HS1 protein is converted into a substrate for c-Fgr by previous Syk-mediated phosphorylation, at site(s) that bind to the SH2 domain of c-Fgr [Ruzzene, M., Brunati, A. M., Marin, O., Donella-Deana, A. & Pinna, L. A. (1996) Biochemistry 35, 5327-5332]. Here we show that a phosphopeptide derived from one such site, HS1-(320-329)-phosphopeptide (PEGDYpEEVLE), enhances up to tenfold, in a dose-dependent manner, the catalytic activity of c-Fgr either assayed with peptide substrates or evaluated as intermolecular autophosphorylation of c-Fgr itself. The dephosphorylated HS1-(320-329)-peptide is totally ineffective, while the stimulatory efficacy of other phosphopeptides derived from the polyoma virus middle T antigen-(393-402) sequence, c-Src, and c-Fgr autophosphorylation sites, and the C-terminal c-Src site (Tyr527) is variable and correlates reasonably well with the predicted affinity for the c-Fgr SH2 domain. Stimulation of c-Fgr catalytic activity is also promoted by the full-length HS1 protein, previously tyrosine phosphorylated by Syk, and is accounted for by an increased Vmax while the Km values are unchanged. If the normal activator of c-Fgr kinase, Mg2+, is replaced by Mn2+, stimulation by HS1-(320-329)-phosphopeptide is still observable with peptide substrates, while autophosphorylation is, in contrast, inhibited by the phosphopeptide. These findings, in conjunction with the ability of previously autophosphorylated c-Fgr to be stimulated by HS1-(320-329)-phosphopeptide, support the view that stimulation of c-Fgr by phosphopeptide is not or is not entirely a consequence of increased autophosphorylation. Interestingly, neither Syk and C-terminal Src kinase nor three other members of the Src family (Lyn, Lck, and Fyn) are susceptible to stimulation by phosphopeptide, as observed with c-Fgr. These data support the notion that c-Fgr undergoes a unique mechanism of activation promoted by tyrosine-phosphorylated polypeptide that binds to its SH2 domain. This suggests that such a mode of regulation is peculiar of protein-tyrosine kinases committed to the secondary phosphorylation of sequentially phosphorylated proteins.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Rat liver Golgi apparatus contains a protein kinase similar to the casein kinase of lactating mammary gland.

By using a beta-casein-derived specific peptide substrate for mammary gland Golgi-enriched-fraction casein kinase, phosphorylating activity has been detected in the Golgi apparatus of rat liver, spleen and to a lesser extent, kidney and brain, while the other post-nuclear cytoplasmic fractions are totally devoid of such a casein kinase activity. In contrast ubiquitous protein kinases CK1 and CK2 (casein kinases 1 and 2), tested with their specific peptide substrates, display different subcellular distribution and are almost undetectable in the Golgi fraction. The absence of CK2 in the Golgi fraction has been also confirmed using specific antibodies. The relatedness between the liver Golgi apparatus casein kinase (G-CK) and the bona fide mammary gland Golgi-enriched-fraction casein kinase (GEF-CK) is supported by a variety of observations, notably: (a) identical peptide substrate specificity, consistent with an S-X-E-X consensus sequence; (b) preference for Mn2+, and, to a lesser extent, Co2+, over Mg2+, as activating cation; (c) superimposable elution profiles from DEAE-Sepharose, heparin-Sepharose, and Superdex 200, this latter consistent with a molecular mass around 500 kDa; (d) insensitivity to staurosporine and heparin (a potent inhibitor of CK2) and inability to use GTP as phosphate donor (by contrast to CK2). These data provide the evidence for the existence of a third class of ubiquitous casein kinases here termed G-CK, distinct from CK1 and CK2, specifically located to the Golgi apparatus and related to the bona fide casein kinase(s) responsible for the phosphorylation of casein secreted from lactating mammary gland. The possible involvement of G-CK in the phosphorylation of secretory pathways proteins at S-X-E motifs is discussed.

Amino Acid Sequence↗

PubMed1997

Evidence for localization of the myotonic dystrophy protein kinase to the terminal cisternae of the sarcoplasmic reticulum.

Myotonic dystrophy is an autosomal dominant multisystem disease primarily affecting skeletal muscle and is characterized by the presence of an amplified trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene. In this study, the subcellular localization of the myotonic dystrophy protein kinase in muscle tissues has been investigated at both morphological and biochemical level, by using antibodies against the myotonic dystrophy protein kinase. Immunofluorescence studies and Western-blot analysis were carried out with antibodies raised against both a synthetic peptide and a recombinant fusion protein fragment specific for the myotonic dystrophy protein kinase. The kinase is localized both to the surface membranes, and within the skeletal fibres in the region of the A-I band boundary. Consistent with the A-I location of the kinase is that Western-blot analysis of purified fractions from sarcoplasmic reticulum show that triads and sarcoplasmic reticulum terminal cisternae are immunoreactive for two myotonic dystrophy protein kinase proteins of different molecular weight (85 and 54 kDa). The relative amount of these two proteins is different in relation to the muscle type, the 85 kDa protein being more evident in skeletal than in cardiac fibres. In addition, immunofluorescence studies of cardiac muscle reveal a heavy concentration of DM-PK localized to the intercalated discs, as well as a weaker reaction in the sarcoplasm. These results taken together suggest that multiple isoforms of the DM-PK may exist and that they may be differentially located in muscle tissues.

Animals↗

PubMed1997

Cartilage associated protein (CASP) is a novel developmentally regulated chick embryo protein.

A subtracted cDNA library was generated to identify cDNAs specific for chondrocyte mRNAs preferentially expressed at the hypertrophic stage with respect to early differentiation stages. The characterization of a cDNA isolated from this library that hybridizes with a 1.8 kb mRNA is described here. This mRNA is expressed at extremely low levels in dedifferentiated chondrocytes cultured in adherent conditions, at very low levels in differentiating chondrocytes and at very high levels in hypertrophic chondrocytes cultured in suspension conditions. In the developing chick embryo this mRNA is detectable in RNAs extracted from several other tissues besides cartilage. The described cDNA contains a complete open reading frame coding for a polypeptide of about 33 kDa. Homology searches with known cDNA and protein sequences have revealed that the chicken protein is related to the amino-terminal half of two mammalian nuclear antigens. By immunohistochemistry with specific rabbit antisera a strong signal was detected in the cartilage extracellular matrix of selected regions of the developing skeleton. Because of this localization of the antigen we named this protein cartilage associated protein (hereafter referred to as CASP).

Amino Acid Sequence↗

PubMed1996

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↗

PubMed1996

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↗