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European Journal of Biochemistry Nov 1993Vitamin-K-dependent protein S is an anticoagulant plasma protein which functions as cofactor to activated protein C (APC) in the degradation of coagulation factors Va...
Vitamin-K-dependent protein S is an anticoagulant plasma protein which functions as cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa. In addition, it interacts with C4b-binding protein (C4BP), a regulator of the complement system. Using a human protein S cDNA clone as probe, cDNA clones for rabbit protein S were isolated from a rabbit liver cDNA library. The cDNA sequence encoded the mature protein S and 12 residues of the leader sequence. The amino acid sequence of the single-chain 634-amino-acid-residue-long rabbit protein S molecule was 82% and 81% identical to those of human and bovine protein S, respectively. Northern blotting demonstrated protein S mRNA not only in liver but also in reproductive organs (testis, ovary and uterus), in lung and brain. Recombinant rabbit protein S was expressed in eucaryotic cells and found to be post-translationally modified, i.e. it had the correct amino terminus, contained N-linked carbohydrate side chains, gamma-carboxyglutamic acid residues and beta-hydroxylated aspartic acid and asparagine residues. Recombinant rabbit protein S bound calcium like its human counterpart, as judged by its migration in the presence of calcium on agarose-gel electrophoresis. Rabbit protein S has been reported to be species specific with respect to its interaction with APC and not to function with bovine APC. However, we found it to act as cofactor to both human and bovine APC, albeit it was somewhat more efficient with human than with bovine APC. Rabbit protein S, like its human and bovine counterparts, bound human C4BP in a reaction which was associated with the loss of its APC-cofactor activity. However, unlike human plasma, rabbit plasma appeared to contain only the free form of protein S as a radiolabeled rabbit protein S tracer added to rabbit plasma migrated as free protein S on agarose-gel electrophoresis. Addition of human C4BP to rabbit plasma resulted in the formation of a C4BP-protein-S complex, suggesting an explanation for the absence of complexed protein S in rabbit plasma to be sought for in the structure of rabbit C4BP.
Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Carrier Proteins; Cloning, Molecular; DNA, Complementary; Humans; Integrin alphaXbeta2; Molecular Sequence Data; Protein S; Rabbits; Recombinant Proteins; Sequence Analysis; Sequence Homology, Amino Acid; Vitamin K
PubMed: 8223642
DOI: 10.1111/j.1432-1033.1993.tb18314.x -
Genetics Mar 2021The protein molecular chaperone Hsp90 (Heat shock protein, 90 kilodalton) plays multiple roles in the biogenesis and regulation of client proteins impacting myriad...
The protein molecular chaperone Hsp90 (Heat shock protein, 90 kilodalton) plays multiple roles in the biogenesis and regulation of client proteins impacting myriad aspects of cellular physiology. Amino acid alterations located throughout Saccharomyces cerevisiae Hsp90 have been shown to result in reduced client activity and temperature-sensitive growth defects. Although some Hsp90 mutants have been shown to affect activity of particular clients more than others, the mechanistic basis of client-specific effects is unknown. We found that Hsp90 mutants that disrupt the early step of Hsp70 and Sti1 interaction, or show reduced ability to adopt the ATP-bound closed conformation characterized by Sba1 and Cpr6 interaction, similarly disrupt activity of three diverse clients, Utp21, Ssl2, and v-src. In contrast, mutants that appear to alter other steps in the folding pathway had more limited effects on client activity. Protein expression profiling provided additional evidence that mutants that alter similar steps in the folding cycle cause similar in vivo consequences. Our characterization of these mutants provides new insight into how Hsp90 and cochaperones identify and interact with diverse clients, information essential for designing pharmaceutical approaches to selectively inhibit Hsp90 function.
Topics: Peptidyl-Prolyl Isomerase F; DNA Helicases; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Heat-Shock Proteins; Molecular Chaperones; Mutation; Nuclear Proteins; Protein Binding; Protein Folding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factor TFIIH
PubMed: 33789348
DOI: 10.1093/genetics/iyab009 -
Molecular and Cellular Biology Nov 2004The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR...
The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.
Topics: Cell Cycle Proteins; Checkpoint Kinase 2; DNA Damage; DNA Replication; DNA, Fungal; DNA-Binding Proteins; Endodeoxyribonucleases; Exodeoxyribonucleases; Fungal Proteins; Genes, Fungal; Intracellular Signaling Peptides and Proteins; Models, Biological; Mutation; Phleomycins; Phosphoproteins; Phosphorylation; Protein Serine-Threonine Kinases; Replication Protein A; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Transcription Factors
PubMed: 15509802
DOI: 10.1128/MCB.24.22.10016-10025.2004 -
The Journal of Biological Chemistry Aug 1993Reversible association of protein S with C4b-binding protein (C4BP) in plasma down-regulates protein S activity, since free protein S but not the protein S.C4BP complex...
Reversible association of protein S with C4b-binding protein (C4BP) in plasma down-regulates protein S activity, since free protein S but not the protein S.C4BP complex is an anticoagulant cofactor for activated protein C. To identify regions on the surface of protein S that mediate complex formation with C4BP, a number of nonoverlapping synthetic pentadecapeptides comprising protein S sequences were prepared and tested for their ability to inhibit complex formation. The most potent pentadecapeptide, residues 420-434 (PSP-420) (SGIKEIIQEKQNKHC), gave half-maximal effect at 20 microM. A peptide with the reverse sequence, 434-420, did not inhibit. A peptide containing the sequence of protein S residues 408-434 inhibited complex formation by > 95% with 50% inhibition at 5 microM peptide. Biotinylated C4BP bound specifically to plates coated with PSP-420 but not with the 434-420 peptide; and biotinylated PSP-420 bound to plates coated with C4BP. Rabbit antibodies were raised against several keyhole limpet hemocyanin-conjugated peptides, and each was tested for ability to inhibit complex formation. Anti-PSP-420 antibody potently inhibited complex formation with half-maximal effect at 25 nM IgG. A monoclonal antibody (LJ-56) made against PSP-420 showed high affinity for protein S and inhibited complex formation; this monoclonal antibody specifically recognized free protein S but not the protein S.C4BP complex. These results imply that the PSP-420 sequence is surface-exposed, capable of binding to C4BP, and essential for protein S binding to C4BP.
Topics: Amino Acid Sequence; Antibodies, Monoclonal; Binding Sites; Carrier Proteins; Complement C4b; Complement Inactivator Proteins; Epitopes; Glycoproteins; Humans; Molecular Sequence Data; Peptide Fragments; Protein Binding; Protein S
PubMed: 7688369
DOI: No ID Found -
Cell Feb 1995We report the identification of ligands for Tyro 3 (alternatively called Sky, rse, brt, or tif) and Axl (alternatively, Ark or UFO), members of a previously orphan...
We report the identification of ligands for Tyro 3 (alternatively called Sky, rse, brt, or tif) and Axl (alternatively, Ark or UFO), members of a previously orphan family of receptor-like tyrosine kinases. These ligands correspond to protein S, a protease regulator that is a potent anticoagulant, and Gas6, a protein related to protein S but lacking any known function. Our results are reminiscent of recent findings that the procoagulant thrombin, a protease that drives clot formation by cleaving fibrinogen to form fibrin, also binds and activates intracellular signaling via a G protein-coupled cell surface receptor. Proteases and protease regulators that also activate specific cell surface receptors may serve to integrate coagulation with associated cellular responses required for tissue repair and growth, as well as to coordinate protease cascades and associated cellular responses in other systems, such as those involved in growth and remodeling of the nervous system.
Topics: 3T3 Cells; Amino Acid Sequence; Animals; Astrocytes; Blotting, Northern; Cell Division; Chromatography, Affinity; Gene Expression Regulation; Intercellular Signaling Peptides and Proteins; Ligands; Mice; Models, Biological; Molecular Sequence Data; Oncogene Proteins; Phosphorylation; Protein Binding; Protein S; Proteins; Proto-Oncogene Proteins; RNA, Messenger; Receptor Protein-Tyrosine Kinases; Recombinant Fusion Proteins; Schwann Cells; Axl Receptor Tyrosine Kinase
PubMed: 7867073
DOI: 10.1016/0092-8674(95)90520-0 -
Eukaryotic Cell Aug 2005The protein kinase C (PKC) pathway is involved in the maintenance of cell shape and cell integrity in Saccharomyces cerevisiae. Here, we show that this pathway mediates...
The Rgd1p Rho GTPase-activating protein and the Mid2p cell wall sensor are required at low pH for protein kinase C pathway activation and cell survival in Saccharomyces cerevisiae.
The protein kinase C (PKC) pathway is involved in the maintenance of cell shape and cell integrity in Saccharomyces cerevisiae. Here, we show that this pathway mediates tolerance to low pH and that the Bck1 and Slt2 proteins belonging to the mitogen-activated protein kinase cascade are essential for cell survival at low pH. The PKC pathway is activated during acidification of the extracellular environment, and this activation depends mainly on the Mid2p cell wall sensor. Rgd1p, which encodes a Rho GTPase-activating protein for the small G proteins Rho3p and Rho4p, also plays a role in low-pH response. The rgd1Delta strain is sensitive to low pH, and Rgd1p activates the PKC pathway in an acidic environment. Inactivation of both genes in the double mutant rgd1Delta mid2Delta strain renders yeast cells unable to survive at low pH as in bck1Delta and slt2Delta strains. Our data provide evidence for the existence of two distinct ways, one involving Mid2p and the other involving Rgd1p, with both converging to the cell integrity pathway to mediate low-pH tolerance in Saccharomyces cerevisiae. Nevertheless, even if Rgd1p acts on the PKC pathway, it seems that its mediating action on low-pH tolerance is not limited to this pathway. As the Mid2p amount plays a role in rgd1Delta sensitivity to low pH, Mid2p seems to act more like a molecular rheostat, controlling the level of PKC pathway activity and thus allowing phenotypical expression of RGD1 inactivation.
Topics: Calcium-Binding Proteins; Cell Survival; Cell Wall; GTPase-Activating Proteins; Genotype; Hydrogen-Ion Concentration; Intracellular Signaling Peptides and Proteins; MADS Domain Proteins; Membrane Glycoproteins; Membrane Proteins; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Models, Biological; Phosphorylation; Protein Kinase C; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Suppression, Genetic; Temperature; Time Factors; Transcription Factors
PubMed: 16087742
DOI: 10.1128/EC.4.8.1375-1386.2005 -
The FEBS Journal Sep 2016Most mitochondrial proteins are synthesized in the cytosol prior to their import into the organelle. It is commonly accepted that cytosolic factors are required for...
Most mitochondrial proteins are synthesized in the cytosol prior to their import into the organelle. It is commonly accepted that cytosolic factors are required for delivering precursor proteins to the mitochondrial surface and for keeping newly synthesized proteins in an import-competent conformation. However, the identity of such factors and their defined contribution to the import process are mostly unknown. Using a presequence-containing model protein and a site-directed photo-crosslinking approach in yeast cells we identified the cytosolic chaperones Hsp70 (Ssa1) and Hsp90 (Hsp82) as well as their cochaperones, Sti1 and Ydj1, as putative cytosolic factors involved in mitochondrial protein import. Deletion of STI1 caused both alterations in mitochondrial morphology and lower steady-state levels of a subset of mitochondrial proteins. In addition, double deletion of STI1 with the mitochondrial import factors, MIM1 or TOM20, showed a synthetic growth phenotype indicating a genetic interaction of STI1 with these genes. Moreover, recombinant cytosolic domains of the import receptors Tom20 and Tom70 were able to bind in vitro Sti1 and other cytosolic factors. In summary, our observations point to a, direct or indirect, role of Sti1 for mitochondrial functionality.
Topics: Adenosine Triphosphatases; Cross-Linking Reagents; Cytosol; Gene Deletion; Genes, Fungal; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Heat-Shock Proteins; Membrane Proteins; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Molecular Chaperones; Organelle Biogenesis; Protein Transport; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 27412066
DOI: 10.1111/febs.13813 -
Molecular and Cellular Biology Jun 1998In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the...
In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlike pho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4. Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner. Mutation of PHO85 suppressed the glycogen storage deficiency of snf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state. Deletion of PCL8 and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1 mutant strain. This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10.
Topics: Cyclin-Dependent Kinases; Cyclins; DNA-Binding Proteins; Fungal Proteins; Glycogen; Glycogen Synthase; Protein Phosphatase 1; Protein Serine-Threonine Kinases; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity; Transcription Factors
PubMed: 9584169
DOI: 10.1128/MCB.18.6.3289 -
The Journal of Clinical Investigation Dec 1990The carboxy-terminal region of protein S has been recently been observed to be involved in the interaction between protein S and C4b-binding protein (Walker, F. J. 1989....
The carboxy-terminal region of protein S has been recently been observed to be involved in the interaction between protein S and C4b-binding protein (Walker, F. J. 1989. J. Biol. Chem. 264:17645-17658). A synthetic peptide, GVQLDLDEAI, corresponding to that region of protein S has been used to investigate the protein S/C4b-binding protein interaction in vitro and in vivo. Rabbit activated protein C possesses species-specific anticoagulant activity for which rabbit protein S functions as a cofactor. In plasma, rabbit protein S is found in complex with C4b-binding protein. GVQLDLDEAI can inhibit this interaction, resulting in enhancement of the anticoagulant activity of rabbit activated protein C. The effect of the peptide can be blocked by the concurrent addition of human or rabbit C4b-binding protein. When infused into rabbits, GVQLDLDEAI was cleared from the circulation with a half-life of 80 min. This is significantly less rapid than the clearance of similarly sized control peptides (half-life of 15 min), but much more than that of bovine protein S, a much larger protein (half-life of 15 h). Plasma samples removed from the rabbits after infusion with GVQLDLDEAI were found to have increased concentrations of free protein S and to show enhanced anticoagulation by rabbit activated protein C ex vivo in a dose-dependent manner. The concentration for half-maximal effect (5 microM) was very similar to that observed in vitro. These results suggest that the formation of a complex between protein S and C4b-binding protein is important in the regulation of protein S activity in vivo, and that modulation of this interaction allows one to influence the anticoagulant activity of the protein C pathway.
Topics: Amino Acid Sequence; Animals; Anticoagulants; Blood Coagulation; Carrier Proteins; Complement Inactivator Proteins; Factor Xa; Glycoproteins; Molecular Sequence Data; Peptides; Protein Binding; Protein C; Protein S; Rabbits; Structure-Activity Relationship
PubMed: 2147696
DOI: 10.1172/JCI114926 -
Proceedings of the National Academy of... Nov 2001The spatial arrangement of COPII coat protein subunits was analyzed by crosslinking to an artificial membrane surface and by electron microscopy of coat proteins and...
The spatial arrangement of COPII coat protein subunits was analyzed by crosslinking to an artificial membrane surface and by electron microscopy of coat proteins and coated vesicle surfaces. The efficiency of COPII subunit crosslinking to phospholipids declined in order of protein recruitment to the coat: Sar1p > Sec23/24p >> Sec13/31p. Deep-etch rotary shadowing and electron microscopy were used to explore the COPII subunit structure with isolated proteins and coated vesicles. Sec23/24 resembles a bow tie, and Sec13/31p contains terminal bilobed globular structures bordering a central rod. The surface structure of COPII vesicles revealed a coat built with polygonal units. The length of the side of the hexagonal/pentagonal units is close to the dimension of the central rod-like segment of Sec13/31. Partially uncoated profiles revealed strands of Sec13/31p stripped from the vesicle surface. We conclude that the coat subunits form layers displaced from the membrane surface in reverse order of addition to the coat.
Topics: COP-Coated Vesicles; Carrier Proteins; Cell Membrane; Fungal Proteins; GTPase-Activating Proteins; Membrane Proteins; Monomeric GTP-Binding Proteins; Nuclear Pore Complex Proteins; Phosphoproteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins
PubMed: 11717432
DOI: 10.1073/pnas.241522198