Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNA aptamers that bind to the Ras-binding domain (RBD) of a proto-oncogene product, Raf-1, were isolated from a pool of random sequences using a glutathione S-transferase-fused RBD (GST-RBD). The RNA molecules bind to the GST-RBD, but not to GST, with dissociation constants of about 300 nM. In contrast, these RNA aptamers do not bind to the Ras-binding domain of the RGL protein, which is also known to be activated by Ras. The aptamers actually compete with Ras for binding to the Raf-1 RBD. The anti-Raf-1 aptamers may be used to specifically inhibit the Ras-Raf interaction in the complicated signaling network in mammalian cells.
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PMID:RNA aptamers that specifically bind to the Ras-binding domain of Raf-1. 988 8

BTF3, initially discovered as a factor required for transcription inititation of RNA polymerase II, is expressed in two isoforms, termed a and b. BTF3b, the transcriptionally inactive isoform, was identified as an interaction partner of protein kinase CK2 subunit beta employing the interaction trap system for screening ofa HeLa cDNA fusion library. We report here on the interaction between the other isoform, BTF3a, and protein kinase CK2. The complete cDNA of BTF3a was cloned by RT-PCR and used for analysis in the two-hybrid system with a three-reporter yeast strain. Interaction of BTF3a with CK2 subunits alpha, alpha' or beta was detectable by one of three reporters, whereas the CK2beta - BTF3a interaction was activating two reporters. It was also shown that BTF3a is phosphorylated in vitro by the alpha2beta2 holoenzyme, but not by alpha or alpha' alone, indicating the requirement of beta for substrate recognition. Immunoprecipitations of GST-fused BTF3a carried out in vitro resulted in co-precipitation of beta. Similarly, GST-BTF3a, but not GST alone isolated with glutathione agarose beads from buffer containing recombinant CK2 subunits was found complexed with alpha and beta, likely representing alpha2beta2 holoenzyme. The data show a weak, nevertheless specific interaction of protein kinase CK2 via subunit beta with the putative transcription factor BTF3a in vitro and in vivo, and a role of BTF3a as a potential new substrate for CK2.
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PMID:BTF3 is a potential new substrate of protein kinase CK2. 1009

The Interferon Regulatory Factors (IRFS) play an important role in the transcriptional control of growth regulatory and immunoregulatory genes. The inducibility and availability of IRF-1 and IRF-2 are influenced by external stimuli, such as virus infection or interferon treatment. In the present study, we sought to examine the potential modulatory role of phosphorylation on IRF-1 transcriptional activity. During the purification of IRF recombinant proteins, a kinase activity copurified with IRF-1 (and IRF-2) from baculovirus infected Sf9 insect cell extracts, but not from E. coli extracts. The kinase activity was also identified in Jurkat T cells, specifically interacted with IRF proteins in GST affinity chromatography, and phosphorylated IRF-1 with high specificity in vitro. Using an in gel kinase assay with recombinant IRF-1 as substrate, two molecular weight forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Furthermore, far western analysis of protein-protein interactions demonstrated that casein kinase II directly interacted with IRF-1 protein. Deletion mutation analysis of IRF-1 revealed that IRF-1 was phosphorylated at two clustered sites, one located between amino acids 138-150, the other in the C-terminal acidic activation domain between amino acids 219-231. Cotransfection studies comparing wild type and point mutated forms of IRF-1 demonstrated that mutations of the four phosphoaceptor residues in the C-terminal transactivation domain, significantly decreased transactivation by IRF-1, indicating that casein kinase II may be involved in the regulation of IRF-1 function. Strikingly, the casein kinase II clusters in IRF-1 resemble the sites identified in the C-terminal PEST domain of IkappaBalpha. The present experiments, together with previously published studies with IkappaBalpha, c-Jun and other proteins, indicate a broad role for casein kinase II phosphorylation in the regulation of transcription factor activity.
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PMID:A role for casein kinase II phosphorylation in the regulation of IRF-1 transcriptional activity. 1009 6

In order to aid in an understanding of the cellular functions of protein kinase CK2, a search for interacting proteins was carried out using a 32P-labeled CK2 overlay method. Several proteins were found to associate with CK2 by this assay; among them, one protein of 110 kDa appeared to be the most prominent one. The possible association of CK2 with p110 was suggested by experiments involving the co-immunoprecipitation using anti-CK2 antibodies. Further analysis using GST-CK2 fusion proteins demonstrated that the CK2-p110 interaction occurred through the CK2alpha/alpha' subunits. To identify p110, it was purified using a GST-CK2 affinity column, and internal amino acid sequencing was then performed. p110 was found to be nucleolin, a nucleolar protein that may be important for rRNA synthesis; a possible role of CK2 in the control of this process is suggested. Using the same CK2 overlay technique, another interacting protein, insulin receptor substrate 1 (IRS-1), was also identified. By applying a modified overlay method using individual 35S-labeled CK2 subunits, obtained by in vitro translation in rabbit reticulate lysates, it was determined that CK2 associates with IRS-1 through its alpha/alpha' subunits; i.e. in keeping with the fact that IRS-1 is a known substrate for CK2. However, further work is needed to examine the association of CK2 with IRS-1 in vivo in order to fully understand the significance of the interaction.
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PMID:Identification of proteins that associate with protein kinase CK2. 1009 12

We have found that the guanine nucleotide exchange factor for ras, Cdc25p, interacts with Ssa1p in Saccharomyces cerevisiae. This interaction was observed with GST-fused Cdc25p polypeptides and confirmed by coimmunoprecipitation with the endogenous Cdc25p. Hsp82 appeared also to be co-immunoprecipitated with Cdc25p, albeit to a lower level than Hsp70. In a strain deleted for SSA1 and SSA2, we observed a reduced cellular content of Cdc25p. Consistent with a reduced activity of the cAMP-dependent PKA pathway, the rate of accumulation of both trehalose and glycogen was stimulated in the ssa-deleted strain. Expression of SSA1 reversed these effects, whereas co-expression of SSA1 and PDE2 restored high accumulation. The expression of genes repressed by cAMP, GAC1 and TPS1, fused to beta-galactosidase, was also stimulated by deletion of SSA genes. The effect of ssa deletion on glycogen accumulation was lost in a strain deleted for CDC25 rescued by the RAS2ile152 allele. Altogether, these results lead to the conclusion that Ssa1p positively controls the cAMP pathway through Cdc25p. We propose that this connection plays a critical role in the adaptation of cells to stress conditions.
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PMID:Ssa1p chaperone interacts with the guanine nucleotide exchange factor of ras Cdc25p and controls the cAMP pathway in Saccharomyces cerevisiae. 1009 33

Dictyostelium expresses at least two proteins of the cyclin-dependent kinase (Cdk) family, Cdc2 and Crp. Cdc2 levels remain relatively constant during differentiation, whereas the levels of Crp increase dramatically as differentiation progresses. Crp is highly related to the mammalian Cdk5, and p25 (a truncated form of p35, the activating subunit of Cdk5 from mammalian brain) stimulates the histone H1 kinase activity of GST-Crp by several fold. In contrast, p25 does not stimulate the histone H1 kinase activity of GST-Cdc2 or the Cdc2 activity present in cell extracts from vegetative Dictyostelium cells. GST-Cdc2, in vitro translated Cdc2 and Cdc2 from all stages of differentiation bind to p13suc1. In contrast, GST-Crp, in vitro translated Crp and the Crp protein present in cell extracts do not bind to p13suc1. We have confirmed a previous report by Arakane and Maeda [J. Plant Res. (1997) 110, 81-85] that there is a peak of p13suc1 bound histone H1 kinase activity during late development, but we found that there was no corresponding peak of p13suc1 bound Cdc2 protein that corresponds to this activity. Taken together, these data suggest that neither Cdc2 nor Crp is responsible for the late developmental peak of histone H1 kinase activity that binds to p13suc1.
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PMID:Binding and catalytic properties of the Cdc2 and Crp proteins of Dictyostelium. 1010 87

Drosophila melanogaster casein kinase II (DmCKII) is composed of catalytic alpha and regulatory beta subunits associated as an alpha2beta2 heterotetramer. Using the two-hybrid system, we have screened a Drosophila embryo cDNA library for proteins that interact with DmCKII alpha. One of the cDNAs encodes a novel beta-like polypeptide, which we designate beta'. In situ hybridization localizes the corresponding gene to 56F1-2, a site distinct from that of both the beta gene and the Stellate family of beta-like sequences. The predicted sequence of beta' is more closely related to the beta subunit of Drosophila and other metazoans than to the Stellate family of proteins, suggesting that it is a second regulatory subunit. In vitro reconstitution studies show that a GST-beta' fusion protein associates with the alpha subunit to generate a tetrameric complex with regulatory properties similar to those of the native alpha2beta2 holoenzyme. The data are consistent with the proposed role of the beta' subunit as an integral component of the holoenzyme.
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PMID:A gene located at 56F1-2 in Drosophila melanogaster encodes a novel metazoan beta-like subunit of casein kinase II. 1032 73

Serum and glucocorticoid-inducible kinase (SGK) is a novel member of the serine/threonine protein kinase family that is transcriptionally regulated. In this study, we have investigated the regulatory mechanisms that control SGK activity. We have established a peptide kinase assay for SGK and present evidence demonstrating that SGK is a component of the phosphoinositide 3 (PI 3)-kinase signaling pathway. Treatment of human embryo kidney 293 cells with insulin, IGF-1 or pervanadate induced a 3- to 12-fold activation of ectopically expressed SGK. Activation was completely abolished by pretreatment of cells with the PI 3-kinase inhibitor, LY294002. Treatment of activated SGK with protein phosphatase 2A in vitro led to kinase inactivation. Consistent with the similarity of SGK to other second-messenger regulated kinases, mutation of putative phosphorylation sites at Thr256 and Ser422 inhibited SGK activation. Cotransfection of PDK1 with SGK caused a 6-fold activation of SGK activity, whereas kinase-dead PDK1 caused no activation. GST-pulldown assays revealed a direct interaction between PDK1 and the catalytic domain of SGK. Treatment of rat mammary tumor cells with serum caused hyperphosphorylation of endogenous SGK, and promoted translocation to the nucleus. Both hyperphosphorylation and nuclear translocation could be inhibited by wortmannin, but not by rapamycin.
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PMID:Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway. 1035 15

Sequence analysis of the C-terminal cytosolic domain of human and mouse polycystin-1 has identified three RxS consensus protein kinase A (PKA) phosphorylation motifs. GST-fusion proteins containing the full-length and truncated C-terminal cytosolic domain of murine polycystin-1 were phosphorylated in vitro by the purified catalytic subunit of PKA. This identified a sequence of 25 amino acids, immediately downstream of a previously identified heterotrimeric G-protein activation sequence, as the major site of PKA phosphorylation. Phosphorylation of wild-type and alanine substituted synthetic peptides containing this motif demonstrated that alanine substitution of serine 4159 largely eliminated phosphorylation. Mutation of this residue in the fusion protein reduced phosphorylation by about 70%, whereas mutation of the other two conserved phosphorylation motifs had little effect. We conclude that serine 4159 is the major site of PKA phosphorylation in the C-terminal cytosolic domain of murine polycystin-1.
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PMID:Identification of the major site of in vitro PKA phosphorylation in the polycystin-1 C-terminal cytosolic domain. 1036 54

Methods for the rapid separation of phosphopeptide isomers (peptides with the same sequence but with phosphates on different residues) were developed using capillary zone electrophoresis with ultraviolet (CZE-UV) detection. Uncoated, cationic and neutral capillaries were used with both acidic and basic peptides. These methods enabled the assay of several protein kinases (mitogen activated protein kinase, protein kinase A, GST-tyrosine kinase) and phosphatases (acid, alkaline, and protein tyrosine phosphatase) and the determination of the sites of phosphorylation and dephosphorylation. Incubations of nonphosphorylated or phosphorylated peptide with kinases or phosphatases took place directly in the instrument's autosampler and were monitored over several hours using CZE-UV.
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PMID:Phosphopeptide isomer separation using capillary zone electrophoresis for the study of protein kinases and phosphatases. 1046 77


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