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European Journal of Biochemistry May 1985Purified Ca2+-dependent and phospholipid-dependent protein kinase (protein kinase C) from bovine brain catalysed the phosphorylation of ribosomal protein S6 when...
Purified Ca2+-dependent and phospholipid-dependent protein kinase (protein kinase C) from bovine brain catalysed the phosphorylation of ribosomal protein S6 when incubated with 40S ribosomal subunits from rat liver or from hamster fibroblasts. The phosphorylation was dependent on Ca2+ and phospholipid, and occurred under ionic conditions similar to those which support protein biosynthesis in vitro. Protein kinase C phosphorylated at least three sites on ribosomal protein S6 when incubated with unphosphorylated ribosomes, and increased the extent of phosphorylation of ribosomes previously phosphorylated predominantly on two sites by cyclic-AMP-dependent protein kinase, converting some molecules to the tetraphosphorylated or pentaphosphorylated form. This indicates that protein kinase C can phosphorylate sites on ribosomal protein S6 other than those phosphorylated by the cyclic-AMP-dependent protein kinase, and this conclusion was confirmed by analysis of tryptic phosphopeptides. These results strengthen the possibility that protein kinase C might be involved in catalysing the multisite phosphorylation of ribosomal protein S6 in certain circumstances in vivo.
Topics: Animals; Catalysis; Cricetinae; Fibroblasts; Liver; Peptides; Phosphorylation; Protein Kinase C; Protein Kinases; Rats; Ribosomal Protein S6; Ribosomal Proteins
PubMed: 3158521
DOI: 10.1111/j.1432-1033.1985.tb08879.x -
Biochemistry Mar 2016The mitogen-activated protein kinase ERK2 is able to elicit a wide range of context-specific responses to distinct stimuli, but the mechanisms underlying this...
The mitogen-activated protein kinase ERK2 is able to elicit a wide range of context-specific responses to distinct stimuli, but the mechanisms underlying this versatility remain in question. Some cellular functions of ERK2 are mediated through regulation of gene expression. In addition to phosphorylating numerous transcriptional regulators, ERK2 is known to associate with chromatin and has been shown to bind oligonucleotides directly. ERK2 is activated by the upstream kinases MEK1/2, which phosphorylate both tyrosine 185 and threonine 183. ERK2 requires phosphorylation on both sites to be fully active. Some additional ERK2 phosphorylation sites have also been reported, including threonine 188. It has been suggested that this phospho form has distinct properties. We detected some ERK2 phosphorylated on T188 in bacterial preparations of ERK2 by mass spectrometry and further demonstrate that phosphomimetic substitution of this ERK2 residue impairs its kinase activity toward well-defined substrates and also affects its DNA binding. We used electrophoretic mobility shift assays with oligonucleotides derived from the insulin gene promoter and other regions to examine effects of phosphorylation and mutations on the binding of ERK2 to DNA. We show that ERK2 can bind oligonucleotides directly. Phosphorylation and mutations alter DNA binding and support the idea that signaling functions may be influenced through an alternate phosphorylation site.
Topics: Animals; Mitogen-Activated Protein Kinase 1; Mutation; Oligonucleotides; Phosphorylation; Protein Binding; Protein Structure, Secondary; Rats
PubMed: 26950759
DOI: 10.1021/acs.biochem.6b00096 -
The Journal of Investigative Dermatology Feb 1983The distribution of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase and its substrate proteins was analyzed using soluble and particulate fractions of pig...
The distribution of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase and its substrate proteins was analyzed using soluble and particulate fractions of pig epidermal homogenates. When histone was used as a substrate for this enzyme reaction, protein kinase activity was distributed almost equally between the soluble and particulate fractions. However, the effect of exogenously added cAMP was confined almost exclusively to the soluble enzyme. Endogenous protein phosphorylation in the absence of exogenous histone was higher in the particulate fraction than in the soluble fraction, but the stimulating effect of cAMP was observed only in the soluble fraction. These results indicate that cAMP-dependent protein kinase is predominantly localized in the soluble fraction and phosphorylates soluble epidermal proteins. The particulate fraction contains protein kinase which is cAMP-independent and phosphorylates particulate-bound proteins as well as histone. Based on these observations, the soluble fraction was incubated with [gamma-32P]-ATP in the presence or absence of cAMP, and phosphorylated protein was analyzed by SDS disc- or slab-gel electrophoresis followed by autoradiography. Among many proteins whose phosphorylation was slightly increased by cAMP, a protein with Mr approximately 45,000 was found which was markedly phosphorylated in the presence of cAMP. Although this protein corresponds to one of the richest proteins in the epidermal soluble fraction, an important physiologic role for this phosphorylation has not been clarified.
Topics: Animals; Cyclic AMP; Kinetics; Molecular Weight; Phosphoproteins; Phosphorylation; Protein Kinases; Proteins; Skin; Solubility; Swine
PubMed: 6296235
DOI: 10.1111/1523-1747.ep12531722 -
The Plant Cell Jun 2020Regulation of grain size is crucial for improving crop yield and is also a basic aspect in developmental biology. However, the genetic and molecular mechanisms...
Regulation of grain size is crucial for improving crop yield and is also a basic aspect in developmental biology. However, the genetic and molecular mechanisms underlying grain size control in crops remain largely unknown despite their central importance. Here, we report that the MEI2-LIKE PROTEIN4 (OML4) encoded by the gene is phosphorylated by GLYCOGEN SYNTHASE KINASE2 (GSK2) and negatively controls grain size and weight in rice (). Loss of function of leads to large and heavy grains, while overexpression of causes small and light grains. OML4 regulates grain size by restricting cell expansion in the spikelet hull. is expressed in developing panicles and grains, and the GFP-OML4 fusion protein is localized in the nuclei. Biochemical analyses show that the GSK2 physically interacts with OML4 and phosphorylates it, thereby possibly influencing the stability of OML4. Genetic analyses support that GSK2 and OML4 act, at least in part, in a common pathway to control grain size in rice. These results reveal the genetic and molecular mechanism of a GSK2-OML4 regulatory module in grain size control, suggesting that this pathway is a suitable target for improving seed size and weight in crops.
Topics: Gene Expression Regulation, Plant; Oryza; Phosphorylation; Plant Proteins
PubMed: 32303659
DOI: 10.1105/tpc.19.00468 -
Biochemistry May 2016T4 polynucleotide kinase is widely used for 5'-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3'-phosphorylation....
T4 polynucleotide kinase is widely used for 5'-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3'-phosphorylation. Here, we report the in vitro selection of deoxyribozymes (DNA enzymes) capable of DNA oligonucleotide 3'-phosphorylation, using a 5'-triphosphorylated RNA transcript (pppRNA) as the phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3'-phosphorylated DNA substrate terminus by 2-methylimidazole activation of the 3'-phosphate (forming 3'-MeImp) and subsequent splint ligation with a 5'-amino DNA oligonucleotide. Competing and precedented DNA-catalyzed reactions were DNA phosphodiester hydrolysis or deglycosylation, each also leading to a 3'-phosphate but at a different nucleotide position within the DNA substrate. One oligonucleotide 3'-kinase deoxyribozyme, obtained from an N40 random pool and named 3'Kin1, can 3'-phosphorylate nearly any DNA oligonucleotide substrate for which the 3'-terminus has the sequence motif 5'-NKR-3', where N denotes any oligonucleotide sequence, K = T or G, and R = A or G. These results establish the viabilty of in vitro selection for identifying DNA enzymes that 3'-phosphorylate DNA oligonucleotides.
Topics: DNA, Catalytic; Oligodeoxyribonucleotides; Phosphorylation
PubMed: 27063020
DOI: 10.1021/acs.biochem.6b00151 -
Current Biology : CB Jan 2018Connections between the protein kinases that function within complex cell polarity networks are poorly understood. Rod-shaped fission yeast cells grow in a highly...
Connections between the protein kinases that function within complex cell polarity networks are poorly understood. Rod-shaped fission yeast cells grow in a highly polarized manner, and genetic screens have identified many protein kinases, including the CaMKK-like Ssp1 and the MARK/PAR-1 family kinase Kin1, that are required for polarized growth and cell shape, but their functional mechanisms and connections have been unknown [1-5]. We found that Ssp1 promotes cell polarity by phosphorylating the activation loop of Kin1. Kin1 regulates cell polarity and cytokinesis through unknown mechanisms [4-7]. We performed a large-scale phosphoproteomic screen and found that Kin1 phosphorylates itself and Pal1 to promote growth at cell tips, and these proteins are interdependent for localization to growing cell tips. Additional Kin1 substrates for cell polarity and cytokinesis (Tea4, Mod5, Cdc15, and Cyk3) were also phosphorylated by a second kinase, the DYRK family member Pom1 [8]. Kin1 and Pom1 were enriched at opposite ends of growing cells, and they phosphorylated largely non-overlapping sites on shared substrates. Combined inhibition of both Kin1and Pom1 led to synthetic defects in their shared substrates Cdc15 and Cyk3, confirming a non-redundant functional connection through shared substrates. These findings uncover a new Ssp1-Kin1 signaling pathway, and define its functional and mechanistic connection with Pom1 signaling for cell polarity and cytokinesis. These kinases are conserved in many eukaryotes including humans, suggesting that similar connections and mechanisms might operate in a broad range of cells.
Topics: Cell Division; Cell Polarity; HSP70 Heat-Shock Proteins; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Signal Transduction
PubMed: 29249658
DOI: 10.1016/j.cub.2017.11.034 -
The EMBO Journal May 1995In higher eukaryotes, the cyclin-dependent kinases (CDKs) are negatively regulated by phosphorylation on threonine 14 (T14) and tyrosine 15 (Y15). In fission yeast, the... (Comparative Study)
Comparative Study
In higher eukaryotes, the cyclin-dependent kinases (CDKs) are negatively regulated by phosphorylation on threonine 14 (T14) and tyrosine 15 (Y15). In fission yeast, the Wee1 and mitosis inhibitory kinase 1 (Mik1) protein kinases phosphorylate Y15 in Cdc2. WEE1Hu is the only known protein kinase that can carry out this inhibitory phosphorylation on Y15 in higher eukaryotes. In the present study, we examined the endogenous products of WEE1Hu in human cells and found that the original WEE1Hu cDNA lacked 214 amino acids at the N-terminus. The predicted full-length protein has weak, but significant, similarity over its entire length with Mik1. Thus, we suggest that 'WEE1Hu' is a Mik1-related protein rather than a Wee1 homologue. When isolated in immunoprecipitates, the endogenous WEE1Hu phosphorylated several cyclin-associated CDKs on Y15. WEE1Hu activity increased during S and G2 phases in parallel with the level of protein. Its activity decreased at M phase when WEE1Hu became transiently hyperphosphorylated. In addition, a decrease in WEE1Hu protein level was observed at M/G1 phase. Apparently, the hyperphosphorylation and degradation in combination caused inactivation of WEE1Hu at M phase and the following G1 phase. These results suggest that the activity of WEE1Hu is regulated by phosphorylation and proteolytic degradation, and that WEE1Hu plays a role in inhibiting mitosis before M phase by phosphorylating cyclin B1-Cdc2.
Topics: Amino Acid Sequence; Base Sequence; Cell Cycle; Cell Cycle Proteins; Cloning, Molecular; Cyclin-Dependent Kinases; DNA, Complementary; HeLa Cells; Humans; Mitosis; Molecular Sequence Data; Nuclear Proteins; Phosphorylation; Protein-Tyrosine Kinases; S Phase; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Sequence Homology, Amino Acid; Species Specificity; Tyrosine
PubMed: 7743995
DOI: 10.1002/j.1460-2075.1995.tb07180.x -
Neurobiology of Disease Nov 2020Leucine-rich repeat kinase 2 (LRRK2), the major causative gene product of autosomal-dominant Parkinson's disease, is a protein kinase that phosphorylates a subset of Rab...
Leucine-rich repeat kinase 2 (LRRK2), the major causative gene product of autosomal-dominant Parkinson's disease, is a protein kinase that phosphorylates a subset of Rab GTPases. Since pathogenic LRRK2 mutations increase its ability to phosphorylate Rab GTPases, elucidating the mechanisms of how Rab phosphorylation is regulated by LRRK2 is of great importance. We have previously reported that chloroquine-induced lysosomal stress facilitates LRRK2 phosphorylation of Rab10 to maintain lysosomal homeostasis. Here we reveal that Rab10 phosphorylation by LRRK2 is potently stimulated by treatment of cells with a set of lysosome stressors and clinically used lysosomotropic drugs. These agents commonly promoted the formation of LRRK2-coated enlarged lysosomes and extracellular release of lysosomal enzyme cathepsin B, the latter being dependent on LRRK2 kinase activity. In contrast to the increase in Rab10 phosphorylation, treatment with lysosomotropic drugs did not increase the enzymatic activity of LRRK2, as monitored by its autophosphorylation at Ser1292 residue, but rather enhanced the molecular proximity between LRRK2 and its substrate Rab GTPases on the cytosolic surface of lysosomes. Lysosomotropic drug-induced upregulation of Rab10 phosphorylation was likely a downstream event of Rab29 (Rab7L1)-mediated enzymatic activation of LRRK2. These results suggest a regulated process of Rab10 phosphorylation by LRRK2 that is associated with lysosomal overload stress, and provide insights into the novel strategies to halt the aberrant upregulation of LRRK2 kinase activity.
Topics: Animals; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Lysosomes; Mice; Phosphorylation; rab GTP-Binding Proteins
PubMed: 32919031
DOI: 10.1016/j.nbd.2020.105081 -
Sheng Li Xue Bao : [Acta Physiologica... Jun 2014Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is the most abundant kinase within excitatory synapses in the mammalian brain. It interacts with and...
Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is the most abundant kinase within excitatory synapses in the mammalian brain. It interacts with and phosphorylates a large number of synaptic proteins, including major ionotropic glutamate receptors (iGluRs) and group I metabotropic glutamate receptors (mGluRs), to constitutively and/or activity-dependently regulate trafficking, subsynaptic localization, and function of the receptors. Among iGluRs, the N-methyl-D-aspartate receptor (NMDAR) is a direct target of CaMKII. By directly binding to an intracellular C-terminal (CT) region of NMDAR GluN2B subunits, CaMKII phosphorylates a serine residue (S1303) in the GluN2B CT. CaMKII also phosphorylates a serine site (S831) in the CT of α-amino-3-hydroxy-5- methylisoxazole-4-propionic acid receptors. This phosphorylation enhances channel conductance and is critical for synaptic plasticity. In addition to iGluRs, CaMKII binds to the proximal CT region of mGluR1a, which enables the kinase to phosphorylate threonine 871. Agonist stimulation of mGluR1a triggers a CaMKII-mediated negative feedback to facilitate endocytosis and desensitization of the receptor. CaMKII also binds to the mGluR5 CT. This binding seems to anchor and accumulate inactive CaMKII at synaptic sites. Active CaMKII dissociates from mGluR5 and may then bind to adjacent GluN2B to mediate the mGluR5-NMDAR coupling. Together, glutamate receptors serve as direct substrates of CaMKII. By phosphorylating these receptors, CaMKII plays a central role in controlling the number and activity of the modified receptors and determining the strength of excitatory synaptic transmission.
Topics: Calcium-Calmodulin-Dependent Protein Kinase Type 2; Neuronal Plasticity; Phosphorylation; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Serine; Synapses; Synaptic Transmission
PubMed: 24964855
DOI: No ID Found -
Proceedings of the National Academy of... Dec 1984Protein kinase C, purified to homogeneity, was found to phosphorylate and activate tyrosine hydroxylase that had been partially purified from pheochromocytoma PC 12...
Protein kinase C, purified to homogeneity, was found to phosphorylate and activate tyrosine hydroxylase that had been partially purified from pheochromocytoma PC 12 cells. These actions of protein kinase C required the presence of calcium and phospholipid. This phosphorylation of tyrosine hydroxylase reduced the Km for the cofactor 6-methyltetrahydropterine from 0.45 mM to 0.11 mM, increased the Ki for dopamine from 4.2 microM to 47.5 microM, and produced no change in the Km for tyrosine. Little or no change in apparent Vmax was observed. These kinetic changes are similar to those seen upon activation of tyrosine hydroxylase by cAMP-dependent protein kinase. Two-dimensional phosphopeptide maps of tyrosine hydroxylase were identical whether the phosphorylation was catalyzed by protein kinase C or by the catalytic subunit of cAMP-dependent protein kinase. Both protein kinases phosphorylated serine residues. The results suggest that protein kinase C and cAMP-dependent protein kinase phosphorylate the same site(s) on tyrosine hydroxylase and activate tyrosine hydroxylase by the same mechanism.
Topics: Animals; Brain; Cattle; Dopamine; Enzyme Activation; Kinetics; Macromolecular Substances; Phosphorylation; Protein Kinase C; Protein Kinases; Tyrosine 3-Monooxygenase
PubMed: 6151178
DOI: 10.1073/pnas.81.24.7713