Two cell lines have been established from insulinomas obtained by targeted expression of the simian virus 40 T antigen gene in transgenic mice. These cell lines, designated MIN6 and MIN7, produce insulin and T antigen and have morphological characteristics of pancreatic beta cells. MIN6 cells exhibit glucose-inducible insulin secretion comparable with cultured normal mouse islet cells, whereas MIN7 cells do not. Both cell lines produce liver-type glucose transporter (GT) mRNA at high level. Brain-type GT mRNA is also present at considerable level in MIN7 cells, but is barely detectable in MIN6 cells, suggesting that exclusive expression of the liver-type GT is related to glucose-inducible insulin secretion. MIN6 cells do not express either major histocompatibility (MHC) class I or class II antigens on the cell surface. However, treatment with interferon-gamma induces high levels of MHC class I antigens, and a combination of interferon-gamma and tumor necrosis factor-alpha induces a MHC class II antigen on the cell surface. These results emphasize that the MIN6 cell line retains physiological characteristics of normal beta cells. The MIN6 cell line will be especially useful to analyze the molecular mechanisms by which beta cells regulate insulin secretion in response to extracellular glucose concentrations. We discuss a possible role of GT isoforms in glucose sensing by beta cells.
We tested the contribution of the small GTPase Rho and its downstream target p160ROCK during the early stages of axon formation in cultured cerebellar granule neurons. p160ROCK inhibition, presumably by reducing the stability of the cortical actin network, triggered immediate outgrowth of membrane ruffles and filopodia, followed by the generation of initial growth cone-ike membrane domains from which axonal processes arose. Furthermore, a potentiation in both the size and the motility of growth cones was evident, though the overall axon elongation rate remained stable. Conversely, overexpression of dominant active forms of Rho or ROCK was suggested to prevent initiation of axon outgrowth. Taken together, our data indicate a novel role for the Rho/ROCK pathway as a gate critical for the initiation of axon outgrowth and the control of growth cone dynamics.
Action polymerization is essential for a variety of cellular processes including movement, cell division and shape change. The induction of actin polymerization requires the generation of free actin filament barbed ends, which results from the severing or uncapping of pre-existing actin filaments [1] [2], or de novo nucleation, initiated by the Arp2/3 complex [3] [4] [5] [6] [7]. Although little is known about the signaling pathways that regulate actin assembly, small GTPases of the Rho family appear to be necessary [8] [9] [10] [11]. In thrombin-stimulated platelets, the Rho family GTPase Rac1 induces actin polymerization by stimulating the uncapping of actin filament barbed ends [2]. The mechanism by which Rac regulates uncapping is unclear, however. We previously demonstrated that Rac interacts with a type I phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) in a GTP-independent manner [12] [13]. Because PIP 5-kinases synthesize phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a lipid that dissociates capping proteins from the barbed ends of actin filaments [14] [15] [16], they are good candidates for mediating the effects of Rac on actin assembly. Here, we have identified the Rac-associated PIP 5-kinase as the PIP 5-kinase isoforms alpha and beta. When added to permeabilized platelets, PIP 5-kinase alpha induced actin filament uncapping and assembly. In contrast, a kinase-inactive PIP 5-kinase alpha mutant failed to induce actin assembly and blocked assembly stimulated by thrombin or Rac. Furthermore, thrombin- or Rac-induced actin polymerization was inhibited by a point mutation in the carboxyl terminus of Rac that disrupts PIP 5-kinase binding. These results demonstrate that PIP 5-kinase alpha is a critical mediator of thrombin- and Rac-dependent actin assembly.
Accumulating evidence suggests that phosphatidylinositol metabolism is essential for membrane traffic in the cell. Of particular importance, phosphatidylinositol transfer protein and the type I phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) have been identified as cytosolic components required for ATP-dependent, Ca 2؉ -activated secretion. In order to identify PI4P5K isoforms that may play important roles in regulated insulin secretion from pancreatic -cells, we employed the polymerase chain reaction with degenerate primers and screening of a cDNA library of the murine pancreatic -cell line MIN6. Two novel cDNAs, designated PI4P5K-I␣ and PI4P5K-I, were identified, which contained complete coding sequences encoding 539-or 546-amino acid proteins, respectively. These cDNAs were expressed in mammalian cells with an adenoviral expression vector. Proteins of both isoforms migrated at 68 kDa on SDS-polyacrylamide gel electrophoresis and exhibited phosphatidylinositol-4-phosphate 5-kinase activity, which was activated by phosphatidic acid, indicating that these proteins were type I isoforms. While these isoforms share a marked amino acid sequence homology in their central portion, the amino-and carboxyl-terminal regions differ significantly. Northern blot analysis depicted that tissue distributions differed between the two isoforms. Molecular identification of type I PI4P5K isoforms in insulin-secreting cells should provide insights into the role of phosphatidylinositol metabolism in regulated exocytosis of insulin-containing large dense core vesicles.Exocytotic release of neurotransmitters and hormones is a highly complex process. In addition to Ca 2ϩ and ATP, regulated fusion of secretory granules with the plasma membrane requires membrane proteins, v-SNARE 1 and t-SNARE. Also required are cytosolic proteins, including N-ethylmaleimide-sensitive factor and soluble N-ethylmaleimide-sensitive factor attachment proteins (reviewed in Ref. 1). Recently, two other cytosolic protein components required for ATP-dependent priming for Ca 2ϩ -activated secretion have been identified: phosphatidylinositol transfer protein (2) and the type I phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) (3). PI4P5K produces, from phosphatidylinositol 4-phosphate (PtdIns(4)P), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), which has been demonstrated to be important in various cellular processes. PtdIns(4,5)P 2 acts as a substrate for phospholipase C, generating the major second messenger molecules, inositol 1,4,5-triphosphate and diacylglycerol (4). In addition, PtdIns-(4,5)P 2 itself has also been demonstrated to function as a regulator molecule in several cellular processes, including actin filament reorganization (5, 6) and exocytosis (7). More recently, it has been postulated that PtdIns(4,5)P 2 functions in the fusion of intracellular vesicles with target membranes (8). Despite these important roles played by the type I PI4P5K, molecular identification of the enzyme has yet to be carried out.Insulin secretion from pancr...
Type I phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of phosphatidylinositol 4,5-bisphosphate, an essential lipid molecule in various cellular processes. Here, we report the cloning of the third member (PIP5K␥) and the characterization of members of the type I PIP5K family. Type I PIP5K␥ has two alternative splicing forms, migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the central portion of this isoform shows approximately 80% identity with those of the ␣ and  isoforms. Northern blot analysis revealed that the ␥ isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the  isoform has the greatest V max value for the PtdIns(4)P kinase activity and the ␥ isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the three isoforms, the minimal kinase core sequence of these isoforms were determined as an approximately 380-amino acid region. In addition, carboxyl-terminal regions of the  and ␥ isoforms were found to confer the greatest V max value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the ␣ isoform is essential for the PtdIns(4)P kinase activity. As was the case with the ␣ isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem. 272, 7578 -7581), overexpression of either the  or the ␥ isoform induced an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induced an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules.Recent advances in cell biology have revealed that phosphoinositide metabolism plays an essential role in various cellular processes. Synthesis and breakdown of certain phosphoinositides at appropriate times and intracellular sites appear to be required for complex regulation of these cellular processes. One of the phosphoinositides, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), 1 is located at an important branchpoint in phosphoinositide metabolism. PtdIns(4,5)P 2 serves as a substrate for phosphoinositide-specific phospholipase C (EC 3
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