Alternative Splicing of Novel Exons of Rat Heart-Type Fructose-6-phosphate 2-Kinase/Fructose-2,6-bisphosphatase Gene

Watanabe, Fusao; Furuya, Eisuke

doi:10.1006/bbrc.2001.4648

Cited by 8 publications

(8 citation statements)

References 21 publications

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“…However, the expression of the four PFK2 genes is not limited to their namesake organ (25, 38). The brain expresses both the brain/placental and heart PFK2 genes (24, 26).…”

Section: Resultsmentioning

confidence: 99%

“…Reverse primer for detection of transcripts containing segment B was 5′-GCG GAC TCC ACA GAC AGA AAG TCA-3′ and for transcripts missing segment B was 5′-TCT TTG CAT CCT CTG ACC TCT CCC-3′, respectively. Primers specific to PFKFB2, heart-type PFK2 mRNA splice variant lacking the AMPK phosphorylation target sequence (MRRNS), were designed using published sequences (25, 26). The forward and reverse primers were 5′-CGT GAC AAG CCA ACT GAA GTG GAG-3′ and 5′-AGG GAT GCT GGC ATG CCA AGC TT-3′, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Fructose-2,6-Bisphosphate Is Lower in Copper Deficient Rat Cerebellum Despite Higher Content of Phosphorylated AMP-Activated Protein Kinase

Gybina

Prohaska

2008

Exp Biol Med (Maywood)

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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 Cudeficient (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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“…However, the expression of the four PFK2 genes is not limited to their namesake organ (25, 38). The brain expresses both the brain/placental and heart PFK2 genes (24, 26).…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Fructose-2,6-Bisphosphate Is Lower in Copper Deficient Rat Cerebellum Despite Higher Content of Phosphorylated AMP-Activated Protein Kinase

Gybina

Prohaska

2008

Exp Biol Med (Maywood)

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“…The heart isoform also has additional phosphorylation sites at the N-terminus (ser-94) and C-terminus (ser-475, ser-483) that can be phosphorylated by protein kinase C, calmodulin-dependent kinases and kinases activated by insulin and growth factor signalling [2]. Various splice variants have been reported for both PFKFB2 and PFKFB3 in human and rat tissue which differ in the C-terminal domain and therefore in their regulatory properties [13][14][15][16].…”

Section: Introductionmentioning

confidence: 99%

A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic β-cells

Arden

Hampson

Huang

et al. 2008

Biochemical Journal

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PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase) catalyses the formation and degradation of fructose 2,6-P(2) (fructose 2,6-bisphosphate) and is also a glucokinase-binding protein. The role of fructose 2,6-P(2) in regulating glucose metabolism and insulin secretion in pancreatic beta-cells is unresolved. We down-regulated the endogenous isoforms of PFK-2/FBPase-2 with siRNA (small interfering RNA) and expressed KA (kinase active) and KD (kinase deficient) variants to distinguish between the role of PFK-2/FBPase-2 protein and the role of its product, fructose 2,6-P(2), in regulating beta-cell function. Human islets expressed the PFKFB2 (the gene encoding isoform 2 of the PFK2/FBPase2 protein) and PFKFB3 (the gene encoding isoform 3 of the PFK2/FBPase2 protein) isoforms and mouse islets expressed PFKFB2 at the mRNA level [RT-PCR (reverse transcription-PCR)]. Rat islets expressed PFKFB2 lacking the C-terminal phosphorylation sites. The glucose-responsive MIN6 and INS1E cell lines expressed PFKFB2 and PFKFB3. PFK-2 activity and the cell content of fructose 2,6-P(2) were increased by elevated glucose concentration and during pharmacological activation of AMPK (AMP-activated protein kinase), which also increased insulin secretion. Partial down-regulation of endogenous PFKFB2 and PFKFB3 in INS1E by siRNA decreased PFK-2/FBPase-2 protein, fructose 2,6-P(2) content, glucokinase activity and glucoseinduced insulin secretion. Selective down-regulation of glucose-induced fructose 2,6-P(2) in the absence of down-regulation of PFK-2/FBPase-2 protein, using a KD PFK-2/FBPase-2 variant, resulted in sustained glycolysis and elevated glucose-induced insulin secretion, indicating an over-riding role of PFK-2/FBPase-2 protein, as distinct from its product fructose 2,6-P(2), in potentiating glucose-induced insulin secretion. Whereas down-regulation of PFK-2/FBPase-2 decreased glucokinase activity, overexpression of PFK-2/FBPase-2 only affected glucokinase distribution. It is concluded that PFK-2/FBPase-2 protein rather than its product fructose 2,6-P(2) is the over-riding determinant of glucose-induced insulin secretion through regulation of glucokinase activity or subcellular targeting.

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“…Isoforms were named according to where they were first discovered (liver, skeletal muscle, foetal, cardiac, testis, brain and placental/ ubiquitous/inducible forms), but their distributions may be wider than these names indicate (Marsin et al ., 2002). At least four genes encode PFK‐2/FBPase‐2 proteins, PFKFB1‐4, with additional diversity generated by expression from different promoters and differential mRNA splicing (Darville et al ., 1991; El‐Maghrabi et al ., 2001; Okar et al ., 2001; Watanabe and Furuya, 2001).…”

Section: Introductionmentioning

confidence: 99%

14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase

Rubio

2003

The EMBO Journal

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The cardiac isoform of 6‐phosphofructo‐2‐kinase/ fructose‐2,6‐bisphosphatase (PFK‐2), regulator of the glycolysis‐stimulating fructose‐2,6‐bisphosphate, was among human HeLa cell proteins that were eluted from a 14‐3‐3 affinity column using the phosphopeptide ARAApSAPA. Tryptic mass fingerprinting and phospho‐specific antibodies showed that Ser466 and Ser483 of 14‐3‐3‐affinity‐purified PFK‐2 were phosphorylated. 14‐3‐3 binding was abolished by selectively dephosphorylating Ser483, and 14‐3‐3 binding was restored when both Ser466 and Ser483 were phosphorylated with PKB, but not when Ser466 alone was phosphorylated by AMPK. Furthermore, the phosphopeptide RNYpS483VGS blocked binding of PFK‐2 to 14‐3‐3s. These data indicate that 14‐3‐3s bind to phosphorylated Ser483. When HeLa cells expressing HA‐tagged PFK‐2 were co‐transfected with active PKB or stimulated with IGF‐1, HA‐PFK‐2 was phosphorylated and bound to 14‐3‐3s. The response to IGF‐1 was abolished by PI 3‐kinase inhibitors. In addition, IGF‐1 promoted the binding of endogenous PFK‐2 to 14‐3‐3s. When cells were transduced with penetratin‐linked AARAApSAPA, we found that this reagent bound specifically to 14‐3‐3s, blocked the IGF‐1‐induced binding of HA‐PFK‐2 to 14‐3‐3s, and completely inhibited the IGF‐1‐induced increase in cellular fructose‐2,6‐bisphosphate. These findings suggest that PKB‐dependent binding of 14‐3‐3s to phospho‐Ser483 of cardiac PFK‐2 mediates the stimulation of glycolysis by growth factor.

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Alternative Splicing of Novel Exons of Rat Heart-Type Fructose-6-phosphate 2-Kinase/Fructose-2,6-bisphosphatase Gene

Cited by 8 publications

References 21 publications

Fructose-2,6-Bisphosphate Is Lower in Copper Deficient Rat Cerebellum Despite Higher Content of Phosphorylated AMP-Activated Protein Kinase

Fructose-2,6-Bisphosphate Is Lower in Copper Deficient Rat Cerebellum Despite Higher Content of Phosphorylated AMP-Activated Protein Kinase

A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic β-cells

14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase

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