scholarly journals Dual requirement for a newly identified phosphorylation site in p70s6k.

1997 ◽  
Vol 17 (9) ◽  
pp. 5648-5655 ◽  
Author(s):  
B A Moser ◽  
P B Dennis ◽  
N Pullen ◽  
R B Pearson ◽  
N A Williamson ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Phosphorylation Site ◽  
Serum Stimulation ◽  
Kinase C ◽  
Homologous Site ◽  
Phosphopeptide Analysis

The activation of p70s6k is associated with multiple phosphorylations at two sets of sites. The first set, S411, S418, T421, and S424, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1. The second set of sites, T229 (in the catalytic domain) and T389 and S404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues. Here we describe the identification and mutational analysis of three new phosphorylation sites, T367, S371, and T447, all of which have a recognition motif similar to that of the first set of sites. A mutation of T367 or T447 to either alanine or glutamic acid had no apparent effect on p70s6k activity, whereas similar mutations of S371 abolished kinase activity. Of these three sites and their surrounding motifs, only S371 is conserved in p70s6k homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371 phosphorylation is regulated by an external mechanism. Phosphopeptide analysis of S371 mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389 phosphorylation, a phosphorylation site previously shown to be essential for kinase activity. Nevertheless, the substitution of an acidic residue at T389, which mimics phosphorylation at this site, did not rescue mutant p70s6k activity, indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity.

Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog

1992 ◽  
Vol 12 (1) ◽  
pp. 172-182
Author(s):  
K S Lee ◽  
D E Levin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Phosphorylation Site ◽  
Cell Lysis ◽  
Amino Acid Identity ◽  
Temperature Sensitive ◽  
Stabilizing Agents ◽  
Kinase C

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth and division. To identify additional components of the pathway in which PKC1 functions, we isolated extragenic suppressors of a pkc1 deletion mutant. All of the suppressor mutations were dominant for suppressor function and defined a single locus, which was designated BCK1 (for bypass of C kinase). A molecular clone of one suppressor allele, BCK1-20, was isolated on a centromere-containing plasmid through its ability to rescue a conditional pkc1 mutant. The BCK1 gene possesses a 4.4-kb uninterrupted open reading frame predicted to encode a 163-kDa protein kinase. The BCK1 gene product is not closely related to any known protein kinase, sharing only 45% amino acid identity with its closest known relative (the STE11-encoded protein kinase) through a region restricted to its putative C-terminal catalytic domain. Deletion of BCK1 resulted in a temperature-sensitive cell lysis defect, which was suppressed by osmotic stabilizing agents. Because pkc1 mutants also display a cell lysis defect, we suggest that PKC1 and BCK1 may normally function within the same pathway. Suppressor alleles of BCK1 differed from the wild-type gene in a region surrounding a potential PKC phosphorylation site immediately upstream of the predicted catalytic domain. This region may serve as a hinge between domains whose interaction is regulated by PKC1.

scholarly journals Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog.

1992 ◽  
Vol 12 (1) ◽  
pp. 172-182 ◽  
Author(s):  
K S Lee ◽  
D E Levin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Phosphorylation Site ◽  
Cell Lysis ◽  
Amino Acid Identity ◽  
Temperature Sensitive ◽  
Stabilizing Agents ◽  
Kinase C

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth and division. To identify additional components of the pathway in which PKC1 functions, we isolated extragenic suppressors of a pkc1 deletion mutant. All of the suppressor mutations were dominant for suppressor function and defined a single locus, which was designated BCK1 (for bypass of C kinase). A molecular clone of one suppressor allele, BCK1-20, was isolated on a centromere-containing plasmid through its ability to rescue a conditional pkc1 mutant. The BCK1 gene possesses a 4.4-kb uninterrupted open reading frame predicted to encode a 163-kDa protein kinase. The BCK1 gene product is not closely related to any known protein kinase, sharing only 45% amino acid identity with its closest known relative (the STE11-encoded protein kinase) through a region restricted to its putative C-terminal catalytic domain. Deletion of BCK1 resulted in a temperature-sensitive cell lysis defect, which was suppressed by osmotic stabilizing agents. Because pkc1 mutants also display a cell lysis defect, we suggest that PKC1 and BCK1 may normally function within the same pathway. Suppressor alleles of BCK1 differed from the wild-type gene in a region surrounding a potential PKC phosphorylation site immediately upstream of the predicted catalytic domain. This region may serve as a hinge between domains whose interaction is regulated by PKC1.

scholarly journals Purification of catalytic domain of rat spleen p72syk kinase and its phosphorylation and activation by protein kinase C

1998 ◽  
Vol 331 (2) ◽  
pp. 649-657 ◽  
Author(s):  
Peter BOROWSKI ◽  
Max HEILAND ◽  
Lutz KORNETZKY ◽  
Sven MEDEM ◽  
Rainer LAUFS
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Assay Method ◽  
Dependent Manner ◽  
Tyrosine Kinase Activity ◽  
Kinase C ◽  
Pkc Isoforms

The catalytic domain of p72syk kinase (CDp72syk) was purified from a 30000 g particulate fraction of rat spleen. The purification procedure employed sequential chromatography on columns of DEAE-Sephacel and Superdex-200, and elution from HA-Ultrogel by chloride. The analysis of the final CDp72syk preparation by SDS/PAGE revealed a major silver-stained 40 kDa protein. The kinase was identified by covalent modification of its ATP-binding site with [14C]5´-fluorosulphonylbenzoyladenosine and by immunoblotting with a polyclonal antibody against the ‘linker ’ region of p72syk. By using poly(Glu4, Tyr1) as a substrate, the specific activity of the enzyme was determined as 18.5 nmol Pi/min per mg. Casein, histones H1 and H2B and myelin basic protein were efficiently phosphorylated by CDp72syk. The kinase exhibited a limited ability to phosphorylate random polymers containing tyrosine residues. CDp72syk autophosphorylation activity was associated with an activation of the kinase towards exogenous substrates. The extent of activation was dependent on the substrates added. CDp72syk was phosphorylated by protein kinase C (PKC) on serine and threonine residues. With a newly developed assay method, we demonstrated that the PKC-mediated phosphorylation had a strong activating effect on the tyrosine kinase activity of CDp72syk. Studies extended to conventional PKC isoforms revealed an isoform-dependent manner (α > βI = βII > γ) of CDp72syk phosphorylation. The different phosphorylation efficiencies of the PKC isoforms closely correlated with the ability to enhance the tyrosine kinase activity.

scholarly journals Identification of Key Phospholipids That Bind and Activate Atypical PKCs

Biomedicines ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 45
Author(s):  
Suresh Velnati ◽  
Sara Centonze ◽  
Federico Girivetto ◽  
Daniela Capello ◽  
Ricardo M. Biondi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Kinase Activity ◽  
Membrane Lipids ◽  
Atypical Protein Kinase C ◽  
Atypical Protein Kinase ◽  
Kinase C ◽  
Specific Regulation ◽  
Phosphatidylinositol 4

PKCζ and PKCι/λ form the atypical protein kinase C subgroup, characterised by a lack of regulation by calcium and the neutral lipid diacylglycerol. To better understand the regulation of these kinases, we systematically explored their interactions with various purified phospholipids using the lipid overlay assays, followed by kinase activity assays to evaluate the lipid effects on their enzymatic activity. We observed that both PKCζ and PKCι interact with phosphatidic acid and phosphatidylserine. Conversely, PKCι is unique in binding also to phosphatidylinositol-monophosphates (e.g., phosphatidylinositol 3-phosphate, 4-phosphate, and 5-phosphate). Moreover, we observed that phosphatidylinositol 4-phosphate specifically activates PKCι, while both isoforms are responsive to phosphatidic acid and phosphatidylserine. Overall, our results suggest that atypical Protein kinase C (PKC) localisation and activity are regulated by membrane lipids distinct from those involved in conventional PKCs and unveil a specific regulation of PKCι by phosphatidylinositol-monophosphates.

scholarly journals Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation.

1988 ◽  
Vol 106 (4) ◽  
pp. 1061-1066 ◽  
Author(s):  
T E McGraw ◽  
K W Dunn ◽  
F R Maxfield
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Phorbol Ester ◽  
Cho Cells ◽  
Transferrin Receptor ◽  
Phosphorylation Site ◽  
Major Protein ◽  
Kinase C ◽  
Recycling Pathway

In Chinese hamster ovary (CHO) fibroblast cells the protein kinase C activating phorbol ester, phorbol myristate acetate (PMA), stimulates an increase in cell surface transferrin receptor (TR) expression by increasing the exocytic rate of the recycling pathway. The human TR expressed in CHO cells is similarly affected by PMA treatment. A mutant human TR in which the major protein kinase C phosphorylation site, serine 24, has been replaced with the non-phosphorylatable amino acid glycine has been constructed to investigate the role of receptor phosphorylation in the PMA induced up-regulation. The Gly-24-substituted receptor binds, internalizes, and recycles Tf. Furthermore, the altered receptor mediates cellular Fe accumulation from diferric-Tf, thereby fulfilling the receptor's major biological role. The Gly-24 TR behaves identically to the wild-type TR when cells are treated with PMA. Therefore, Ser-24 phosphorylation is not required for the PMA-induced redistribution of the human TR expressed in CHO cells. The increased TR expression on the cell surface after PMA treatment results from an increase in the rate of exocytosis of the recycling receptors. No change in the endocytic rate or the size of the recycling receptor pool was observed. These results indicate that the PMA effect on the TR surface expression may result from a more general perturbation of membrane trafficking rather than a specific modulation of the TR.

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