Baltrusch S, Lenzen S, Okar D A, Lange A J, Tiedge M
Institute of Clinical Biochemistry, Hannover Medical School, 30623 Hannover, Germany.
J Biol Chem. 2001 Nov 23;276(47):43915-23. doi: 10.1074/jbc.M105470200. Epub 2001 Aug 24.
The low affinity glucose-phosphorylating enzyme glucokinase shows the phenomenon of intracellular translocation in beta cells of the pancreas and the liver. To identify potential binding partners of glucokinase by a systematic strategy, human beta cell glucokinase was screened by a 12-mer random peptide library displayed by the M13 phage. This panning procedure revealed two consensus motifs with a high binding affinity for glucokinase. The first consensus motif, LSAXXVAG, corresponded to the glucokinase regulatory protein of the liver. The second consensus motif, SLKVWT, showed a complete homology to the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which acts as a key regulator of glucose metabolism. Through yeast two-hybrid analysis it became evident that the binding of glucokinase to PFK-2/FBPase-2 is conferred by the bisphosphatase domain, whereas the kinase domain is responsible for dimerization. 5'-Rapid amplification of cDNA ends analysis and Northern blot analysis revealed that rat pancreatic islets express the brain isoform of PFK-2/FBPase-2. A minor portion of the islet PFK-2/FBPase-2 cDNA clones comprised a novel splice variant with 8 additional amino acids in the kinase domain. The binding of the islet/brain PFK-2/ FBPase-2 isoform to glucokinase was comparable with that of the liver isoform. The interaction between glucokinase and PFK-2/FBPase-2 may provide the rationale for recent observations of a fructose-2,6-bisphosphate level-dependent partial channeling of glycolytic intermediates between glucokinase and glycolytic enzymes. In pancreatic beta cells this interaction may have a regulatory function for the metabolic stimulus-secretion coupling. Changes in fructose-2,6-bisphosphate levels and modulation of PFK-2/FBPase-2 activities may participate in the physiological regulation of glucokinase-mediated glucose-induced insulin secretion.
低亲和力葡萄糖磷酸化酶葡萄糖激酶在胰腺β细胞和肝脏中表现出细胞内易位现象。为了通过系统策略鉴定葡萄糖激酶的潜在结合伴侣，利用M13噬菌体展示的12聚体随机肽库对人β细胞葡萄糖激酶进行筛选。该淘选过程揭示了两个对葡萄糖激酶具有高结合亲和力的共有基序。第一个共有基序LSAXXVAG与肝脏中的葡萄糖激酶调节蛋白相对应。第二个共有基序SLKVWT与双功能酶6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶（PFK-2/FBPase-2）完全同源，该酶是葡萄糖代谢的关键调节因子。通过酵母双杂交分析发现，葡萄糖激酶与PFK-2/FBPase-2的结合由双磷酸酶结构域介导，而激酶结构域负责二聚化。5'-cDNA末端快速扩增分析和Northern印迹分析表明，大鼠胰岛表达PFK-2/FBPase-2的脑型同工型。一小部分胰岛PFK-2/FBPase-2 cDNA克隆包含一种新的剪接变体，其激酶结构域中有8个额外的氨基酸。胰岛/脑型PFK-2/FBPase-2同工型与葡萄糖激酶的结合与肝脏同工型相当。葡萄糖激酶与PFK-2/FBPase-2之间可能为最近观察到的果糖-2,6-二磷酸水平依赖性糖酵解中间产物在葡萄糖激酶和糖酵解酶之间的部分通道化提供了理论依据。在胰腺β细胞中，这种相互作用可能对代谢刺激-分泌偶联具有调节作用。果糖-2,6-二磷酸水平的变化和PFK-2/FBPase-2活性的调节可能参与葡萄糖激酶介导的葡萄糖诱导胰岛素分泌的生理调节。
