Biomedical subjects

L A Pinna

Publications and source records attributed to L A Pinna.

At least 37 records · Page 2Linked to original sources

PubMed2000

Unique features of HIV-1 Rev protein phosphorylation by protein kinase CK2 ('casein kinase-2').

The HIV-1 Rev transactivator is phosphorylated in vitro by protein kinase CK2 at two residues, Ser-5 and Ser-8; these sites are also phosphorylated in vivo. Here we show that the mechanism by which CK2 phosphorylates Rev is unique in several respects, notably: (i) it is fully dependent on the regulatory, beta-subunit of CK2; (ii) it relies on the integrity of an acidic stretch of CK2 beta which down-regulates the phosphorylation of other substrates; (iii) it is inhibited in a dose-dependent manner by polyamines and other polycationic effectors that normally stimulate CK2 activity. In contrast, a peptide corresponding to the amino-terminal 26 amino acids of Rev, including the phosphoacceptor site, is readily phosphorylated by the catalytic subunit of CK2 even in the absence of the beta-subunit. These data, in conjunction with the observation that two functionally inactive derivatives of Rev with mutations in its helix-loop-helix motif are refractory to phosphorylation, indicate the phosphorylation of Rev by CK2 relies on conformational features of distinct regions that are also required for the transactivator's biological activity.

Amino Acid Sequence↗

PubMed2000

Sequential phosphorylation of protein band 3 by Syk and Lyn tyrosine kinases in intact human erythrocytes: identification of primary and secondary phosphorylation sites.

Treatment of intact human erythrocytes with pervanadate induces Tyr (Y)-phosphorylation of the transmembrane protein band 3; in parallel, the activity of the immunoprecipitated tyrosine kinases Syk and Lyn is increased. When erythrocytes are incubated with pervanadate together with PP1, a specific inhibitor of Src kinases, including Lyn, the Y-phosphorylation of band 3 is only partially reduced. Indeed, the PP1-resistant phosphorylation of band 3 precedes and is a prerequisite for its coimmunoprecipitation with Lyn, which interacts with the phosphoprotein via the SH2 domain of the enzyme, as proven by binding competition experiments. Upon recruitment to primarily phosphorylated band 3, Lyn catalyzes the secondary phosphorylation of the transmembrane protein. These data are consistent with the view that band 3 is phosphorylated in intact erythrocytes by both PP1-resistant (most likely Syk) and PP1-inhibited (most likely Lyn) tyrosine kinases according to a sequential phosphorylation process. Similar radiolabeled peptide maps are obtained by tryptic digestion of (32)P-band 3 isolated from either pervanadate-treated erythrocytes or red cell membranes incubated with exogenous Syk and Lyn. It has also been demonstrated by means of mass spectrometry that the primary phosphorylation of band 3 occurs at Y8 and Y21, while the secondary phosphorylation affects Y359 and Y904. (Blood. 2000;96:1550-1557)

Anion Exchange Protein 1, Erythrocyte↗

PubMed2000

Susceptibility of the prion protein to enzymic phosphorylation.

Ten protein kinases have been assayed for their ability to phosphorylate in vitro the recombinant bovine PrP (25-242) (rbPrP). Substantial phosphorylation was observed with PKC, CK2, and two tyrosine kinases, Lyn and c-Fgr. With regard to CK2, phosphorylation occurs at Ser 154 with a stoichiometry of about 0.1 mol phosphate/mol rbPrP, which is doubled by mild heat treatment of rbPrP. Heat also reduces the overall protein ellipticity, suggesting that reversibly unfolded conformers are more susceptible to phosphorylation. Our data disclose the possibility that phosphorylation might modulate PrP biological activity.

Animals↗

PubMed2000

GRP94 (endoplasmin) co-purifies with and is phosphorylated by Golgi apparatus casein kinase.

A phosphorylatable protein band of about 94 kDa (as judged by SDS-PAGE) which co-purifies and co-immunoprecipitates with Golgi apparatus casein kinase (G-CK) from rat lactating mammary gland has been shown by mass spectrometric sequence analysis to be identical or very similar to the glucose-regulated protein, GRP94. GRP94 is also readily phosphorylated by G-CK (K(m)=0.2 microM) at seryl sites which are different from the sites affected by casein kinase-2 (CK2) in the same protein. A study with peptide substrates would indicate that the G-CK sites in GRP94 conform to the motif S-R/K-E-X (X being different from D and E) which is not recognized by CK2.

Amino Acid Motifs↗

PubMed2000

pCMB treatment reveals the essential role of cysteinyl residues in conferring functional competence to the regulatory subunit of protein kinase CK2.

To assess the functional role of the four conserved cysteinyl residues in the regulatory beta-subunit of protein kinase CK2, the effect of pCMB and other reagents of sulfhydryl groups has been investigated. The pCMB-treated beta-subunit has lost its ability to form either homodimers or regular alpha(2)beta(2) heterotetramers with the catalytic subunit. It also fails to increase catalytic activity toward peptide substrates and to mediate the stimulatory effect of polylysine. The pCMB-treated beta-subunit, however, is still able to prevent calmodulin phosphorylation and to physically interact with the alpha-subunit to form inactive complexes whose sedimentation coefficient is lower than that of CK2 holoenzyme. These inactive complexes upon treatment with reducing agents like DTT are converted into a fully active heterotetrameric holoenzyme.

Animals↗

PubMed2000

Ser/Thr phosphorylation of hematopoietic specific protein 1 (HS1): implication of protein kinase CK2.

Hematopoietic lineage cell-specific protein 1 (HS1), a tyrosine multiphosphorylated protein implicated in receptor-mediated apoptosis and proliferative responses, is shown here to become Ser/Thr phosphorylated upon incubation of platelets with radiolabeled inorganic phosphate. The in vivo Ser/Thr phosphorylation of HS1 is enhanced by okadaic acid and reduced by specific inhibitors of casein kinase (CK)2. In vitro, HS1 is an excellent substrate for either CK2 alpha subunit alone (Km = 47 nM) or CK2 holoenzyme, tested in the presence of polylysine (Km = 400 nM). Phosphorylation reaches a stoichiometry of about 2 mol phosphate per mol HS1 and occurs mainly at threonyl residue(s), mostly located in the N-terminal region, but also at seryl residue(s) residing in the central core of the molecule (208-402), as judged from experiments with deleted forms of HS1. Ser/Thr phosphorylation of HS1, either induced in vivo by okadaic acid or catalysed in vitro by CK2, potentiates subsequent phosphorylation at tyrosyl residues. These data indicate the possibility that regulation of HS1 may also be under the control of Ser/Thr phosphorylation, and suggest that in quiescent cells CK2 could play a role in inducing constitutive Tyr phosphorylation of HS1 in the absence of stimuli that activate the protein tyrosine kinase pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme.

The crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides, presents a molecular twofold axis, with each peptide interacting with both alpha chains. In the derived model of the holoenzyme, the regulatory subunits are positioned on the opposite side with respect to the opening of the catalytic sites, that remain accessible to substrates and cosubstrates. The beta subunit can influence the catalytic activity both directly and by promoting the formation of the alpha2 dimer, in which each alpha chain interacts with the active site of the other. Furthermore, the two active sites are so close in space that they can simultaneously bind and phosphorylate two phosphoacceptor residues of the same substrate.

Amino Acid Sequence↗

PubMed2000

Identification of a domain in human immunodeficiency virus type 1 rev that is required for functional activity and modulates association with subnuclear compartments containing splicing factor SC35.

The activity of human immunodeficiency virus Rev as a regulator of viral mRNA expression is tightly linked to its ability to shuttle between the nucleus and cytoplasm; these properties are conferred by a leucine-rich nuclear export signal (NES) and by an arginine-rich nuclear localization signal/RNA binding domain (NLS/RBD) required for binding to the Rev-responsive element (RRE) located on viral unspliced and singly spliced mRNAs. Structure predictions and biophysical measurements indicate that Rev consists of an unstructured region followed by a helix-loop-helix motif containing the NLS/RBD and sequences directing multimerization and by a carboxy-terminal tail containing the NES. We present evidence that the loop portion of the helix-loop-helix region is an essential functional determinant that is required for binding to the RRE and for correct intracellular routing. Data obtained using a protein kinase CK2 phosphorylation assay indicated that the loop region is essential for juxtaposition of helices 1 and 2 and phosphorylation by protein kinase CK2. Deletion of the loop resulted in partial accumulation of Rev in SC35-positive nuclear bodies that resembled nuclear bodies that form in response to inhibition of transcription. Accumulation of the DeltaLoop mutant in nuclear bodies depended on the presence of an intact NES, suggesting that both the loop and the NES play a role in controlling intranuclear compartmentalization of Rev and its association with splicing factors.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Hematopoietic lineage cell specific protein 1 associates with and down-regulates protein kinase CK2.

The catalytic (alpha) subunit of protein kinase CK2 and the hematopoietic specific protein 1 (HS1) display opposite effects on Ha-ras induced fibroblast transformation, by enhancing and counteracting it, respectively. Here we show the occurrence of physical association between HS1 and CK2alpha as judged from both far Western blot and plasmon resonance (BIAcore) analysis. Association of HS1 with CK2alpha is drastically reduced by the deletion of the HS1 C-terminal region (403-486) containing an SH3 domain. HS1, but not its deletion mutant HS1 Delta324-393, lacking a sequence similar to an acidic stretch of the regulatory beta-subunit of CK2, inhibits calmodulin phosphorylation by CK2alpha. These data indicate that HS1 physically interacts with CK2alpha and down-regulates its activity by a mechanism similar to the beta-subunit.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Tyrosine versus serine/threonine phosphorylation by protein kinase casein kinase-2. A study with peptide substrates derived from immunophilin Fpr3.

Protein kinase casein kinase-2 (CK2) is a spontaneously active, ubiquitous, and pleiotropic enzyme that phosphorylates seryl/threonyl residues specified by multiple negatively charged side chains, the one at position n + 3 being of crucial importance (minimum consensus S/T-x-x-E/D/S(P)/T(P). Recently CK2 has been reported to catalyze phosphorylation of the yeast nucleolar immunophilin Fpr3 at a tyrosyl residue (Tyr(184)) fulfilling the consensus sequence of Ser/Thr substrates (Wilson, L.K., Dhillon, N., Thorner, J., and Martin, G.S. (1997) J. Biol. Chem. 272, 12961-12967). Here we show that, by contrast to other tyrosyl peptides fulfilling the consensus sequence for CK2, a peptide reproducing the sequence around Fpr3 Tyr(184) (DEDADIY(184)DEEDYDL) is phosphorylated by CK2, albeit with much higher K(m) (384 versus 4. 3 microM) and lower V(max) (8.4 versus 1,132 nmol.min(-1).mg(-1)) than its derivative with Tyr(184) replaced by serine. The replacement of Asp at position n + 1 with alanine and, to a lesser extent, of Ile at n - 1 with Asp are especially detrimental to tyrosine phosphorylation as compared with serine phosphorylation, which is actually stimulated by the Ile to Asp modification. In contrast the replacement of Glu at n + 3 with alanine almost suppresses serine phosphorylation but not tyrosine phosphorylation. It can be concluded that CK2 is capable to phosphorylate, under special circumstances, tyrosyl residues, which are specified by structural features partially different from those that optimize Ser/Thr phosphorylation.

Amino Acid Sequence↗

PubMed1999

Phosphorylated seryl and threonyl, but not tyrosyl, residues are efficient specificity determinants for GSK-3beta and Shaggy.

Glycogen synthase kinase-3 is involved in diverse functions including insulin signalling and development. In a number of substrates, phosphorylation by glycogen synthase kinase-3 is known to require prior phosphorylation at a Ser in the +4 position relative to its own phosphorylation site. Here we have used synthetic peptides derived from a putative glycogen synthase kinase-3 site in the Drosophila translation initiation factor eIF2B epsilon to investigate the efficacy of residues other than Ser(P) as priming residues for glycogen synthase kinase-3beta and its Drosophila homologue Shaggy. Glycogen synthase kinase-3beta phosphorylated peptides with Ser(P) and Thr(P) in the priming position, but peptides with Tyr(P), Thr, Glu or Asp were not phosphorylated. The Vmax for the Thr(P) peptide was three times higher than that of the Ser(P) peptide. These data suggest that glycogen synthase kinase-3 is unique among phosphate-directed kinases. The priming site specificity of Shaggy is similar to that of mammalian glycogen synthase kinase-3beta. This unpredicted efficacy of Thr(P) in the priming position suggests that there may be other unidentified substrates for these kinases.

Amino Acid Sequence↗

PubMed1999

Structural features underlying the unusual mode of calmodulin phosphorylation by protein kinase CK2: A study with synthetic calmodulin fragments.

To shed light on the paradoxical behaviour of calmodulin, whose phosphorylation is inhibited by the regulatory beta-subunit of protein kinase CK2, a series of peptides encompassing the phosphoacceptor sites of calmodulin have been synthesized and assayed as substrates of CK2 alpha-subunit either alone or combined with the beta-subunit. The shortest peptide whose phosphorylation is reduced instead of being enhanced by the beta-subunit encompasses the sequence 68-106, including the central helix and the Ca2+-binding loop-III. In contrast, the phosphorylation of a peptide encompassing loop II and the central helix (54-92) is stimulated, like that of several shorter peptides, by the beta-subunit. Our data localize to the C-terminal domain of calmodulin the structural elements that are responsible for inverted susceptibility to beta-subunit regulation.

Amino Acid Sequence↗

PubMed1999

Molecular features underlying the sequential phosphorylation of HS1 protein and its association with c-Fgr protein-tyrosine kinase.

The hematopoietic lineage cell-specific protein HS1 was shown to undergo a process of sequential phosphorylation both in vitro and in vivo, which is synergistically mediated by Syk and Src family protein-tyrosine kinases and essential for B cell antigen receptor-mediated apoptosis. We have now identified tyrosine 222 as the HS1 residue phosphorylated by the Src family protein kinases c-Fgr and Lyn, and we show that a truncated form of HS1 (HS1-208-401) lacking the N-terminal putative DNA binding region and the C-terminal Src homology 3 (SH3) domain is still able to undergo all the steps of sequential phosphorylation as efficiently as full-length HS1. We also show that a stable association of phospho-HS1 with c-Fgr through its SH2 domain requires previous autophosphorylation of the kinase and is prevented by subsequent phosphorylation of Tyr-222. Kinetic studies with HS1 and its truncated forms previously phosphorylated by Syk and with a peptide substrate reproducing the sequence around tyrosine 222 support the view that efficient phosphorylation of HS1 by Src family protein kinases entirely relies on TyrP-SH2 domain interaction with negligible, if any, contribution of local specificity determinants. Our data indicate that the proline-rich region of HS1 bordered by tyrosyl residues affected by Syk and Src family kinases represents a functional domain designed to undergo a process of sequential phosphorylation.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Optimal sequences for non-phosphate-directed phosphorylation by protein kinase CK1 (casein kinase-1)--a re-evaluation.

A variety of synthetic peptides derived from either the inhibitor-2 (I-2) phosphoacceptor sites or the optimal sequences selected in an oriented peptide library have been compared for their susceptibility to phosphorylation by protein kinase CK1 (also termed casein kinase-1). The I-2-derived peptides are by far preferred over the library peptides by both rat liver CK1 (and by the alpha/beta, gamma and delta/epsilon isoforms immunoprecipitated from it) and recombinant Xenopus laevis CK1 alpha. The superiority of the I-2-derived peptides over the library ones is reflected by Vmax values one to two orders of magnitude higher while the Km values are comparable. Individual substitutions of any of the aspartic acids with alanine in the I-2-derived peptide RRKHAAIGDDDDAYSITA is detrimental, producing both a fall in Vmax and an increase in Km which are more pronounced at position n -3, but also quite significant at positions n -4, n -5 and, to a lesser extent, n -6. The unfavourable effect of these substitutions is more evident with rat liver CK1 than with recombinant Xenopus laevis CK1 alpha. The chimeric peptide IGDDDDAY-S-IIIFFA, resulting from the combination of the N-terminal acidic sequence of the I-2 (Ser86) site and the C-terminal hydrophobic cluster selected in the library peptides (MAEFDTG-S-IIIFFAKKK and MAYYDAA-S-IIIFFAKKK) is phosphorylated as efficiently as the I-2-derived peptide in terms of both Km and Vmax. These combined data strongly support the conclusion that, at variance with the optimal sequences selected in the library, optimal non-phosphate-directed phosphorylation of peptide substrates by CK1 critically relies on the presence of a cluster of acidic residues (preferably aspartic acid) upstream from position n -2, while the highly hydrophobic region downstream from serine selected in the library appears to be dispensable. The reason for these discrepancies remains unclear. The possibility that the library data are biased by the invariant elements forming its scaffold (MA-x-x-x-x-x-SI-x-x-x-x-AKKK) would be consistent with the observation that the library-selected peptides, despite their low Km values, fail to compete against the phosphorylation of protein and peptide substrates by CK1, suggesting that they bind to elements partially distinct from those responsible for substrate recognition.

Amino Acid Sequence↗

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

Retractions.

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Retraction Notice↗