Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report the identification of a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase gene (PFKFB3) isolated from a human fetal brain cDNA library. The gene was localized to 10p15-->p14 by fluorescence in situ hybridization. The entire cDNA (4,322 bp) codes for a polypeptide of 520 amino acid residues (molecular weight, 59.571 kDa). Structural analysis showed the presence of a kinase domain located at the amino terminus and a bisphosphatase domain at the carboxy terminus, characteristic of previously described 6-phosphofructo-2-kinase/fructose 2, 6-bisphosphatase isozymes. In addition, a phosphorylation site for cAMP-dependent protein kinase was found at the carboxy terminus. Northern blot analysis showed the presence of a unique 4.8-kb mRNA expressed in the different tissues studied. In mammalian COS-1 cells, this cDNA drives the expression of an active isozyme. Taken together, these results identify the presence of a gene coding for a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase isozyme which is ubiquitously expressed.
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PMID:Molecular cloning, expression, and chromosomal localization of a ubiquitously expressed human 6-phosphofructo-2-kinase/ fructose-2, 6-bisphosphatase gene (PFKFB3). 1007 80

Previous studies have shown that (i) the insulin-induced activation of heart 6-phosphofructo-2-kinase (PFK-2) is wortmannin-sensitive, but is insensitive to rapamycin, suggesting the involvement of phosphatidylinositol 3-kinase; and (ii) protein kinase B (PKB) activates PFK-2 in vitro by phosphorylating Ser-466 and Ser-483. In this work, we have studied the effects of phosphorylation of these residues on PFK-2 activity by replacing each or both residues with glutamate. Mutation of Ser-466 increased the V(max) of PFK-2, whereas mutation of Ser-483 decreased citrate inhibition. Mutation of both residues was required to decrease the K(m) for fructose 6-phosphate. We also studied the insulin-induced activation of heart PFK-2 in transfection experiments performed in human embryonic kidney 293 cells. Insulin activated transfected PFK-2 by phosphorylating Ser-466 and Ser-483. Kinase-dead (KD) PKB and KD 3-phosphoinositide-dependent kinase-1 (PDK-1) cotransfectants acted as dominant negatives because both prevented the insulin-induced activation of PKB as well as the inactivation of glycogen-synthase kinase-3, an established substrate of PKB. However, the insulin-induced activation of PFK-2 was prevented only by KD PDK-1, but not by KD PKB. These results indicate that the insulin-induced activation of heart PFK-2 is mediated by a PDK-1-activated protein kinase other than PKB.
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PMID:Heart 6-phosphofructo-2-kinase activation by insulin results from Ser-466 and Ser-483 phosphorylation and requires 3-phosphoinositide-dependent kinase-1, but not protein kinase B. 1052 87

A wortmannin-sensitive and insulin-stimulated protein kinase (WISK), which phosphorylates and activates cardiac 6-phosphofructo-2-kinase (PFK-2), was partially purified from perfused rat hearts. Immunoblotting showed that WISK was devoid of protein kinase B (PKB), serum- and glucocorticoid-regulated protein kinase and protein kinase Czeta (PKCzeta). Comparison of the inhibition of WISK, PKCalpha and PKCzeta by different protein kinase inhibitors suggested that WISK was not a member of the PKC family. In addition, WISK contained no detectable phosphoinositide-dependent protein kinase-1 (PDK1) activity. WISK phosphorylated recombinant heart PFK-2 in a time-dependent manner to the extent of 0.4 mol of phosphate incorporated/mol of enzyme subunit, and increased the V(max) of PFK-2 twofold, without affecting the K(m) for fructose 6-phosphate. WISK phosphorylated Ser-466 to a greater extent than Ser-483 in recombinant heart PFK-2, and both sites were demonstrated to be phosphorylated to the same extent by PKB. Gel filtration and in-gel kinase analysis indicated that WISK was a monomer with a M(r) of 56500. Treatment of WISK with protein phosphatase 2A (PP2A) catalytic subunits reversed the effect of insulin, suggesting the involvement of an upstream activating kinase. Indeed, PDK1 was able to partially reactivate the PP2A-treated WISK and this reactivation was not enhanced by PtdIns(3,4,5)P(3)-containing vesicles. Moreover, a single 57000-M(r) band was labelled on incubation of the dephosphorylated WISK preparation with PDK1 and [gamma-(32)P]ATP. These findings provide evidence for the existence of a new protein kinase in the insulin signalling pathway, probably downstream of PDK1.
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PMID:Partial purification and characterization of a wortmannin-sensitive and insulin-stimulated protein kinase that activates heart 6-phosphofructo-2-kinase. 1072 32

Phosphorylation of yeast 6-phosphofructo-2-kinase and its role for the regulation of glycolysis under hypoosmotic conditions were investigated. 6-Phosphofructo-2-kinase was found to be phosphorylated in vitro by protein kinase C at serine 652 and thereby inactivated. Protein phosphatase 2A reversed the phosphorylative inhibition of the enzyme. When yeast cells were shifted to hypotonic media, 6-phosphofructo-2-kinase was found to be phosphorylated and inactivated. Under in vivo conditions, two phosphate residues were incorporated into the enzyme. One of them is bound to serine 652, indicating that this modification was probably caused by yeast protein kinase C1. The second phosphate is bound to Ser8 within the N-terminal peptide T(1-41) which contains several serine residues but no protein kinase C recognition sequence. Site-directed mutagenesis confirmed that the phosphorylation of serine 652 but not the N-terminal modification is responsible for the in vivo inactivation of 6-phosphofructo-2-kinase. The obtained results suggest that the phosphorylation of 6-phosphofructo-2-kinase mediates a response of the cells to an activation of the hypoosmolarity MAP kinase pathway. Via a suppression of glycolysis, the inactivation of 6-phosphofructo-2-kinase is expected to be responsible for the observed accumulation of glucose 6-phosphate, an essential precursor of the cell wall glucans, and the decrease of glycerol, an important osmolyte.
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PMID:Phosphorylation and inactivation of yeast 6-phosphofructo-2-kinase contribute to the regulation of glycolysis under hypotonic stress. 1172 81

Increasing heart workload stimulates glycolysis by enhancing glucose transport and fructose-2,6-bisphosphate (Fru-2,6-P(2)), the latter resulting from 6-phosphofructo-2-kinase (PFK-2) activation. Here, we investigated whether adenosine monophosphate (AMP)-activated protein kinase (AMPK) mediates PFK-2 activation in hearts submitted to increased workload. When heart work was increased, PFK-2 activity, Fru-2,6-P(2) content and glycolysis increased, whereas the AMP:adenosine triphosphate (ATP) and phosphocreatine/creatine (PCr:Cr) ratios, and AMPK activity remained unchanged. Wortmannin, the well-known phosphatidylinositol-3-kinase inhibitor, blocked the activation of protein kinase B and the increase in glycolysis and Fru-2,6-P(2) content induced by increased work. Therefore, the control of heart glycolysis by contraction differs from that in skeletal muscle where AMPK is involved.
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PMID:The stimulation of heart glycolysis by increased workload does not require AMP-activated protein kinase but a wortmannin-sensitive mechanism. 1241 35

In response to changes in the environment, yeast cells coordinate intracellular activities to optimize survival and proliferation. The transductions of diverse extracellular stimuli are exerted through multiple mitogen-activated protein kinase (MAPK) cascades. The high osmolarity glycerol (HOG) MAPK pathway is activated by increased environmental osmolarity and results in a rise of the cellular glycerol concentration to adapt the intracellular osmotic pressure. We studied the importance of the short time regulation of glycolysis under hyperosmotic stress for the survival and proliferation of yeast cells. A stimulation of the HOG-MAPK pathway by increasing the medium osmolarity through addition of salt or glucose to cultivated yeast leads to an activation of 6-phosphofructo-2-kinase (PFK2), which is accompanied by a complex phosphorylation pattern of the enzyme. An increase in medium osmolarity with 5% NaCl activates PFK2 3-fold over the initial value. This change in the activity is the result of a 4-fold phosphorylation of the enzyme mediated by protein kinases from the HOG-MAPK pathway. In the case of hyperosmolar glucose a 5-fold PFK2 activation was achieved by a single phosphorylation with protein kinase A near the carboxyl terminus of the protein on Ser(644) and an additional 5-fold phosphorylation within the same amino-terminal fragment as in the presence of salt. The effect of hyperosmolar glucose is the result of an activation of the Ras-cAMP pathway together with the HOG-MAPK pathway. The activation of PFK2 leads to an activation of the upper part of glycolysis, which is a precondition for glycerol accumulation. Yeast cells containing PFK2 accumulate three times more glycerol than cells lacking PFK2, which are not able to grow under hypertonic stress.
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PMID:High osmolarity glycerol (HOG) pathway-induced phosphorylation and activation of 6-phosphofructo-2-kinase are essential for glycerol accumulation and yeast cell proliferation under hyperosmotic stress. 1503 28

Fru-2,6-P2 (fructose 2,6-bisphosphate) is a signal molecule that controls glycolysis. Since its discovery more than 20 years ago, inroads have been made towards the understanding of the structure-function relationships in PFK-2 (6-phosphofructo-2-kinase)/FBPase-2 (fructose-2,6-bisphosphatase), the homodimeric bifunctional enzyme that catalyses the synthesis and degradation of Fru-2,6-P2. The FBPase-2 domain of the enzyme subunit bears sequence, mechanistic and structural similarity to the histidine phosphatase family of enzymes. The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo-1-kinase), but this proved not to be correct. Molecular modelling of the PFK-2 domain revealed that, instead, it has the same fold as adenylate kinase. This was confirmed by X-ray crystallography. A PFK-2/FBPase-2 sequence in the genome of one prokaryote, the proteobacterium Desulfovibrio desulfuricans, could be the result of horizontal gene transfer from a eukaryote distantly related to all other organisms, possibly a protist. This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with possibly only one of the domains active), indicates that fusion of genes initially coding for separate PFK-2 and FBPase-2 domains might have occurred early in evolution. In the enzyme homodimer, the PFK-2 domains come together in a head-to-head like fashion, whereas the FBPase-2 domains can function as monomers. There are four PFK-2/FBPase-2 isoenzymes in mammals, each coded by a different gene that expresses several isoforms of each isoenzyme. In these genes, regulatory sequences have been identified which account for their long-term control by hormones and tissue-specific transcription factors. One of these, HNF-6 (hepatocyte nuclear factor-6), was discovered in this way. As to short-term control, the liver isoenzyme is phosphorylated at the N-terminus, adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein kinase), leading to PFK-2 inactivation and FBPase-2 activation. In contrast, the heart isoenzyme is phosphorylated at the C-terminus by several protein kinases in different signalling pathways, resulting in PFK-2 activation.
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PMID:6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis. 1517 Mar 86

A wortmannin-sensitive and insulin-stimulated protein kinase (WISK) that phosphorylates and activates heart 6-phosphofructo-2-kinase (PFK-2) was purified from serum-fed HeLa cells and found to contain protein kinase Czeta (PKCzeta). Both WISK and recombinant PKCzeta were inhibited by a pseudo-substrate peptide inhibitor of PKCzeta. WISK and PKCzeta phosphorylated and activated recombinant heart PFK-2 by increasing its Vmax. The phosphorylation sites in heart PFK-2 for WISK were Ser466 and Thr475, whereas PKCzeta phosphorylated only Thr475. In perfused rat hearts, insulin activated protein kinase B (PKB) 16-fold compared with the untreated controls. However in the same experiments, no change in phosphorylation state of the activation loop Thr410 residue of PKCzeta was observed. By contrast, in incubations of isolated rat epididymal adipocytes, where insulin activated PKB 30-fold compared with the untreated controls, a 50% increase in PKCzeta Thr410 phosphorylation was detected. Lastly in HEK 293T cells transfected with heart PFK-2, co-transfection with a kinase-inactive PKCzeta construct failed to prevent insulin-induced PFK-2 activation. Therefore, it is unlikely that PKCzeta is required for PFK-2 activation by insulin in heart.
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PMID:Evaluation of the role of protein kinase Czeta in insulin-induced heart 6-phosphofructo-2-kinase activation. 1682 26

Limitation in copper (Cu) leads to pathophysiology in developing brain. Cu deficiency impairs brain mitochondria and results in high brain lactate suggesting augmented anaerobic glycolysis. AMP activated protein kinase (AMPK) is a cellular energy "master-switch" that is thought to augment glycolysis through phosphorylation and activation phosphofructokinase 2 (PFK2) resulting in increases of the glycolytic stimulator fructose-2,6-bisphosphate (F2,6BP). Previously, Cu deficiency has been shown to augment cerebellar AMPK activation. Cerebella of Cu-adequate (Cu+) and Cu-deficient (Cu-) rat pups were assessed to evaluate if AMPK activation in Cu- cerebella functioned to enhance PFK2 activation and increase F2,BP concentration. Higher levels of pAMPK were detected in Cu- cerebella. However, PFK2 activity, mRNA, and protein abundance were not affected by Cu deficiency. Surprisingly, F2,6BP levels were markedly lower in Cu- cerebella. Lower F2,6BP may be due to inhibition of PFK2 by citrate, as citrate concentration was significantly higher in Cu- cerebella. Data suggest AMPK activation in Cu- cerebellum does not augment glycolysis through a PFK2 mechanism. Furthermore, other metabolite data suggest that glycolysis may actually be blunted, since levels of glucose and glucose-6-phosphate were higher in Cu- cerebella than controls.
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PMID:Fructose-2,6-bisphosphate is lower in copper deficient rat cerebellum despite higher content of phosphorylated AMP-activated protein kinase. 1870 56

The regulation of metabolism and growth must be tightly coupled to guarantee the efficient use of energy and anabolic substrates throughout the cell cycle. Fructose 2,6-bisphosphate (Fru-2,6-BP) is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in glycolysis. The concentration of Fru-2,6-BP in mammalian cells is set by four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4), which interconvert fructose 6-phosphate and Fru-2,6-BP. The relative functions of the PFKFB3 and PFKFB4 enzymes are of particular interest because they are activated in human cancers and increased by mitogens and low oxygen. We examined the cellular localization of PFKFB3 and PFKFB4 and unexpectedly found that whereas PFKFB4 localized to the cytoplasm (i.e. the site of glycolysis), PFKFB3 localized to the nucleus. We then overexpressed PFKFB3 and observed no change in glucose metabolism but rather a marked increase in cell proliferation. These effects on proliferation were completely abrogated by mutating either the active site or nuclear localization residues of PFKFB3, demonstrating a requirement for nuclear delivery of Fru-2,6-BP. Using protein array analyses, we then found that ectopic expression of PFKFB3 increased the expression of several key cell cycle proteins, including cyclin-dependent kinase (Cdk)-1, Cdc25C, and cyclin D3 and decreased the expression of the cell cycle inhibitor p27, a universal inhibitor of Cdk-1 and the cell cycle. We also observed that the addition of Fru-2,6-BP to HeLa cell lysates increased the phosphorylation of the Cdk-specific Thr-187 site of p27. Taken together, these observations demonstrate an unexpected role for PFKFB3 in nuclear signaling and indicate that Fru-2,6-BP may couple the activation of glucose metabolism with cell proliferation.
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PMID:Nuclear targeting of 6-phosphofructo-2-kinase (PFKFB3) increases proliferation via cyclin-dependent kinases. 1947 63


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