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• W2048787560 abstract "Cyclin-dependent kinase 5 (Cdk5) is widely expressed although kinase activity has been described preferentially in neuronal systems. Cdk5 has an impact on actin polymerization during neuronal migration and neurite outgrowth and deregulation of the kinase has been implicated in the promotion of neurodegeneration. Recently it was shown that Cdk5 modulates dopamine signaling in neurons by regulating DARPP-32 function. In addition, Cdk5 phosphorylates munc-18 and synapsin I, two essential components of the exocytotic machinery. We have shown by reverse transcriptase-polymerase chain reaction, immunocytochemistry, and Western blotting that Cdk5 is present in the insulin-secreting pancreatic β-cell. Subcellular fractionation of isolated β-cells revealed a glucose-induced translocation of membrane-bound Cdk5 protein to lower density fractions. Inhibition of Cdk5 with roscovitine reduced insulin secretion with ∼35% compared with control after glucose stimulation and with ∼65% after depolarization with glucose and KCl. Capacitance measurements performed on single β-cells that expressed a dominant-negative Cdk5 mutant showed impaired exocytosis. The effect on exocytosis by Cdk5 appeared to be independent of changes in free cytoplasmic Ca2+ concentration. Taken together these results show that Cdk5 is present in β-cells and acts as a positive regulator of insulin exocytosis. Cyclin-dependent kinase 5 (Cdk5) is widely expressed although kinase activity has been described preferentially in neuronal systems. Cdk5 has an impact on actin polymerization during neuronal migration and neurite outgrowth and deregulation of the kinase has been implicated in the promotion of neurodegeneration. Recently it was shown that Cdk5 modulates dopamine signaling in neurons by regulating DARPP-32 function. In addition, Cdk5 phosphorylates munc-18 and synapsin I, two essential components of the exocytotic machinery. We have shown by reverse transcriptase-polymerase chain reaction, immunocytochemistry, and Western blotting that Cdk5 is present in the insulin-secreting pancreatic β-cell. Subcellular fractionation of isolated β-cells revealed a glucose-induced translocation of membrane-bound Cdk5 protein to lower density fractions. Inhibition of Cdk5 with roscovitine reduced insulin secretion with ∼35% compared with control after glucose stimulation and with ∼65% after depolarization with glucose and KCl. Capacitance measurements performed on single β-cells that expressed a dominant-negative Cdk5 mutant showed impaired exocytosis. The effect on exocytosis by Cdk5 appeared to be independent of changes in free cytoplasmic Ca2+ concentration. Taken together these results show that Cdk5 is present in β-cells and acts as a positive regulator of insulin exocytosis. N-ethylmaleimide-sensitive factor soluble NSF attachment proteins solubleN-ethylmaleimide-sensitive attachment protein receptor synaptosomal-associated protein of 25 kDa cyclin-dependent kinase dopamine and adenosine 3′,5′-monophosphate-regulated phosphoprotein of 32 kDa dimethyl sulfoxide reverse transcribed-polymerase chain reaction protein phosphatase 1 dominant negative enhanced green fluorescent protein intracellular [Ca2+] Insulin is stored in secretory granules in pancreatic β-cells and upon stimulation with secretagogues insulin is released by exocytosis. Exocytosis has been suggested to be mediated by the same core fusion machinery that traverses intracellular membrane traffic in all cells (1Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1574) Google Scholar, 2Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2611) Google Scholar, 3Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2001) Google Scholar). It was reported that the membrane fusion event required the N -ethylmaleimidesensitive factor (NSF),1 andsoluble NSF AttachmentProteins, α-, β-, and γ-SNAP (1Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1574) Google Scholar, 2Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2611) Google Scholar). In addition to NSF and SNAPs, a 7 S core complex with SNApreceptors or “SNARE” proteins corresponding to the vesicle component synaptobrevin/vesicular-associated membrane protein, as well as the plasma membrane proteins SNAP-25 (synaptosomal-associated protein of 25 kDa) and syntaxin were necessary for neuronal exocytosis (3Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2001) Google Scholar). The SNARE hypothesis proposes that the SNARE proteins form trans-complexes between adjacent membranes, thereby forcing them to proximity. After association of α-SNAP, the ATPase NSF completes the reaction by disassembling the SNARE complex leading to membrane bilayer mixing (3Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2001) Google Scholar). More recently, trans-SNARE pairing and NSF activity has been suggested to act either prior to docking of vesicles or after membrane bilayer mixing (4Ungermann C. Sato K. Wickner W. Nature. 1998; 396: 543-548Crossref PubMed Scopus (279) Google Scholar, 5Xu T. Ashery U. Burgoyne R.D. Neher E. EMBO J. 1999; 18: 3293-3304Crossref PubMed Scopus (90) Google Scholar, 6Xu T. Rammner B. Margittai M. Artalejo A.R. Neher E. Jahn R. Cell. 1999; 99: 713-722Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 7Jahn R. Südhof T.C. Annu. Rev. Biochem. 1999; 68: 863-911Crossref PubMed Scopus (1014) Google Scholar, 8Lin R.C. Scheller R.H. Annu. Rev. Cell Dev. Biol. 2000; 16: 19-49Crossref PubMed Scopus (418) Google Scholar, 9Peters C. Bayer M.J. Bühler S. Andersen J.S. Mann M. Mayer A. Nature. 2001; 409: 581-588Crossref PubMed Scopus (421) Google Scholar). Initially the SNARE proteins were regarded as neuron-specific. However, syntaxin, SNAP-25, synaptobrevin/vesicular-associated membrane protein, and other synaptic proteins regulating neuronal exocytosis have also been identified in pancreatic β-cells, suggesting that the mechanism for insulin secretion is similar to that of neurotransmitter release from synaptic vesicles in neurons (10Jacobsson G. Bean A.J. Scheller R.H. Juntti-Berggren L. Deeney J.T. Berggren P.O. Meister B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12487-12491Crossref PubMed Scopus (194) Google Scholar, 11Sadoul K. Lang J. Montecucco C. Weller U. Regazzi R. Catsicas S. Wollheim C.B. Halban P.A. J. Cell Biol. 1995; 128: 1019-1028Crossref PubMed Scopus (231) Google Scholar, 12Wheeler M.B. Sheu L. Ghai M. Bouquillon A. Grondin G. Weller U. Beaudoin A.R. Bennett M.K. Trimble W.S. Gaisano H.Y. Endocrinology. 1996; 137: 1340-1348Crossref PubMed Scopus (182) Google Scholar, 13Yang S.N. Larsson O. Bränström R. Bertorello A.M. Leibiger B. Leibiger I.B. Moede T. Köhler M. Meister B. Berggren P.O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10164-10169Crossref PubMed Scopus (122) Google Scholar). Regulated secretion of insulin from pancreatic β-cells has to be tightly controlled to ensure glucose homeostasis in the body. The capacity of the β-cell to respond to stimulation is determined by several different signaling pathways. One important feature of these pathways is the phosphorylation cascade of a wide range of cellular substrates. Many different kinases such as protein kinase C, cAMP-dependent protein kinase (protein kinase A), Ca2+/calmodulin-dependent kinase II, mitogen-activated protein kinases, and protein-tyrosine kinases are present in the islets (14Jones P.M. Persaud S.J. Endocr. Rev. 1998; 19: 429-461Crossref PubMed Google Scholar). From neuronal systems it is known that protein phosphorylation of synaptic proteins plays significant roles in modulating different steps during exocytosis and synaptic vesicle recycling (15Turner K.M. Burgoyne R.D. Morgan A. Trends Neurosci. 1999; 22: 459-464Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Presumably, protein kinases are also important players in the managing of secretory granules in the pancreatic β-cell. However, data regarding the physiological relevance for a particular kinase in the regulation of insulin secretion is often contradictory (reviewed in Ref. 14Jones P.M. Persaud S.J. Endocr. Rev. 1998; 19: 429-461Crossref PubMed Google Scholar). Cyclin-dependent kinases (Cdks) and their activator molecules (cyclins) are heterodimers known to be powerful regulators of cell division progression (16Hunter T. Pines J. Cell. 1994; 79: 573-582Abstract Full Text PDF PubMed Scopus (2150) Google Scholar, 17Lees E. Curr. Opin. Cell Biol. 1995; 7: 773-780Crossref PubMed Scopus (265) Google Scholar, 18Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2925) Google Scholar). Although Cdk5 is expressed in most mammalian tissues and interacts with the widespread D-type cyclins, this association apparently does not result in an active kinase (19Lee M.H. Nikolic M. Baptista C.A. Lai E. Tsai L.H. Massague J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3259-3263Crossref PubMed Scopus (116) Google Scholar,20Xiong Y. Zhang H. Beach D. Cell. 1992; 71: 505-514Abstract Full Text PDF PubMed Scopus (899) Google Scholar). The recognition of Cdk5 activity in post-mitotic neurons in the central nervous system suggested functions unrelated to cell cycle control (reviewed in Ref. 21Lew J. Wang J.H. Trends Biochem. Sci. 1995; 20: 33-37Abstract Full Text PDF PubMed Scopus (186) Google Scholar). Neuronal Cdk5 activation was found to be mediated by complex formation with proteins exhibiting only limited homology to classical cyclins (22Ishiguro K. Kobayashi S. Omori A. Takamatsu M. Yonekura S. Anzai K. Imahori K. Uchida T. FEBS Lett. 1994; 342: 203-208Crossref PubMed Scopus (148) Google Scholar, 23Lew J. Huang Q.Q. Qi Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (539) Google Scholar, 24Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (808) Google Scholar, 25Tang D. Yeung J. Lee K.Y. Matsushita M. Matsui H. Tomizawa K. Hatase O. Wang J.H. J. Biol. Chem. 1995; 270: 26897-26903Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Cdk5 is a proline-directed serine/threonine kinase and phosphorylates neurofilaments (26Lew J. Winkfein R.J. Paudel H.K. Wang J.H. J. Biol. Chem. 1992; 267: 25922-25926Abstract Full Text PDF PubMed Google Scholar, 27Hisanaga S. Uchiyama M. Hosoi T. Yamada K. Honma N. Ishiguro K. Uchida T. Dahl D. Ohsumi K. Kishimoto T. Cell Motil. Cytoskeleton. 1995; 31: 283-297Crossref PubMed Scopus (45) Google Scholar, 28Pant A.C. Veeranna Pant H.C. Amin N. Brain Res. 1997; 765: 259-266Crossref PubMed Scopus (59) Google Scholar) and the microtubule-associated protein Tau (22Ishiguro K. Kobayashi S. Omori A. Takamatsu M. Yonekura S. Anzai K. Imahori K. Uchida T. FEBS Lett. 1994; 342: 203-208Crossref PubMed Scopus (148) Google Scholar, 29Kobayashi S. Ishiguro K. Omori A. Takamatsu M. Arioka M. Imahori K. Uchida T. FEBS Lett. 1993; 335: 171-175Crossref PubMed Scopus (219) Google Scholar). It has been suggested that deregulation of Cdk5 results in hyperphosphorylation of Tau, a prominent trait found in post-mortem brains of patients with Alzheimer's disease (30Patrick G.N. Zukerberg L. Nikolic M. de la M.S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1310) Google Scholar). Furthermore, it has been reported that Cdk5 promotes neurite extension of cultured cortical neurons and other mechanisms associated with actin polymerization (31Nikolic M. Dudek H. Kwon Y.T. Ramos Y.F. Tsai L.H. Genes Dev. 1996; 10: 816-825Crossref PubMed Scopus (529) Google Scholar, 32Nikolic M. Chou M.M. Lu W. Mayer B.J. Tsai L.H. Nature. 1998; 395: 194-198Crossref PubMed Scopus (350) Google Scholar, 33Humbert S. Dhavan R. Tsai L. J. Cell Sci. 2000; 113: 975-983Crossref PubMed Google Scholar, 34Veeranna G.J. Shetty K.T. Takahashi M. Grant P. Pant H.C. Brain Res. Mol. Brain Res. 2000; 76: 229-236Crossref PubMed Scopus (74) Google Scholar). A targeted disruption of the gene for Cdk5 results in abnormal brain development and perinatal death (35Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (806) Google Scholar). Lately, both synapsin I and munc-18 have been reported to be substrates for Cdk5, suggesting that Cdk5 might be a regulator of neuronal exocytosis (36Matsubara M. Kusubata M. Ishiguro K. Uchida T. Titani K. Taniguchi H. J. Biol. Chem. 1996; 271: 21108-21113Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 37Shuang R. Zhang L. Fletcher A. Groblewski G.E. Pevsner J. Stuenkel E.L. J. Biol. Chem. 1998; 273: 4957-4966Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 38Fletcher A.I. Shuang R. Giovannucci D.R. Zhang L. Bittner M.A. Stuenkel E.L. J. Biol. Chem. 1999; 274: 4027-4035Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 39de Vries K.J. Geijtenbeek A. Brian E.C. de Graan P.N. Ghijsen W.E. Verhage M. Eur. J. Neurosci. 2000; 12: 385-390Crossref PubMed Scopus (65) Google Scholar). Interestingly, it was also recently reported that Cdk5 modulates dopamine signaling by phosphorylating dopamine and adenosine 3′,5′-monophosphate-regulated phosphoprotein of 32 kDa (DARPP-32) (40Bibb J.A. Snyder G.L. Nishi A. Yan Z. Meijer L. Fienberg A.A. Tsai L.H. Kwon Y.T. Girault J.A. Czernik A.J. Huganir R.L. Hemmings Jr., H.C. Nairn A.C. Greengard P. Nature. 1999; 402: 669-671Crossref PubMed Scopus (489) Google Scholar, 41Nishi A. Bibb J.A. Snyder G.L. Higashi H. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12840-12845Crossref PubMed Scopus (200) Google Scholar). In the present study we report that Cdk5 is expressed outside the nervous system in the insulin-secreting pancreatic β-cell and that this kinase positively affects insulin exocytosis. Expression constructs encoding wild-type (wt) and a dominant-negative (dn) form of Cdk5 were gifts of S. van den Heuvel (42van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (970) Google Scholar). In the dn Cdk5 mutant an Asp is changed to an Asn at amino acid 144. This amino acid substitution results in a catalytic inactive kinase (24Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (808) Google Scholar, 31Nikolic M. Dudek H. Kwon Y.T. Ramos Y.F. Tsai L.H. Genes Dev. 1996; 10: 816-825Crossref PubMed Scopus (529) Google Scholar). Antibodies used were a rabbit polyclonal anti-Cdk5 antibody, C8 (Santa Cruz Biotechnology), a mouse monoclonal anti-insulin antibody, HB125 (Biogenex), a mouse monoclonal anti-syntaxin 1 antibody, HPC-1 (Sigma), and a mouse monoclonal anti-α-tubulin antibody, DM 1A (Sigma). Secondary antibodies used for immunocytochemistry were Texas Red goat anti-mouse IgG (H+L) conjugate purchased from Molecular Probes and fluorescein anti-rabbit IgG (H+L) conjugate purchased from Vector Laboratories. The secondary antibodies used in Western blotting were horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulins (Dako Corp.). Roscovitine was obtained from Calbiochem, diluted to a 10 mm stock in dimethyl sulfoxide (Me2SO), and used in a final concentration of 10 μm. Fura-2/acetoxymetyl ester (Fura-2/AM) was obtained from Molecular Probes and used in a final concentration of 2 μm. Mouse pancreatic β-cells were isolated from 10–12-month-old non-inbred obese hyperglycemic mice (gene ob/ob) as described previously (43Lernmark A. Diabetologia. 1974; 10: 431-438Crossref PubMed Scopus (288) Google Scholar,44Nilsson T. Arkhammar P. Hallberg A. Hellman B. Berggren P.O. Biochem. J. 1987; 248: 329-336Crossref PubMed Scopus (67) Google Scholar). Ob/ob islets are composed of more than 90% β-cells (44Nilsson T. Arkhammar P. Hallberg A. Hellman B. Berggren P.O. Biochem. J. 1987; 248: 329-336Crossref PubMed Scopus (67) Google Scholar, 45Hellman B. Ann. N. Y. Acad. Sci. 1965; 131: 541-558Crossref PubMed Scopus (303) Google Scholar). The cells were seeded onto coverslips and cultured in RPMI 1640 culture medium containing 11 mm glucose supplemented with 10% (v/v) fetal calf serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine. Total RNA was isolated from mouse brain (C57BL6) and pancreatic islets (ob/ob) using the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen). Reverse transcribed-polymerase chain reaction (RT-PCR) was performed using the SuperScriptTM RT-PCR System (Life Technologies, Inc.). Cdk5-specific primers were designed and synthesized (GenSet) according to data base sequences (5′-ACTGTGTTCAAGGCTAAAAACC-3′and 5′-CAATTTCAACTCCCCATTCC-3). The RT-PCR reactions were performed using 10 pmol of each primer and the following program: 30 min at 50 °C and 2 min at 94 °C, followed by 40 cycles of denaturing at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min, ending with a final extension at 72 °C for 7 min. The amplified PCR products were separated by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining. 1-Kilobase DNA ladder (Life Technologies, Inc.) was used as size marker. Primary β-cells from ob/obmice were cultured 2–3 days as described above. The cells were fixed, permeabilized, and stained as described previously (46Andersson J. Fried G. Lilja L. Meister B. Bark C. Eur. J. Cell Biol. 2000; 79: 781-789Crossref PubMed Scopus (9) Google Scholar). The stainings were analyzed with a laser scanning confocal microscope (Leica TCS NT), equipped with a krypton/argon laser. The images were processed with Adobe Photoshop 5.0 software. Pancreatic islets isolated from ob/ob mice and brain tissue from C57BL6 were homogenized in buffer containing (in mm): 20 HEPES, 2 EDTA, 1 MgCl2, 1 phenylmethylsulfonyl fluoride and protease inhibitor mixture according to instructions from the manufacturer (Roche Molecular Diagnostics), pH 7.4. Postnuclear supernatants were ultracentrifuged at 100,000 × g for 40 min to separate soluble and membrane fractions. Protein concentrations were determined using BCA protein assay reagent (Pierce) or Bio-Rad protein assay (Bio-Rad). Samples were separated on 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). Membranes were blocked in phosphate-buffered saline containing 3% blotto (Amersham Pharmacia Biotech) and 0.025% Tween 20 (Merck Eurolab) for 1 h and then probed with primary antibodies overnight at 4 °C. After washing, membranes were incubated with horseradish peroxidase-conjugated immunoglobulins for 45 min at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence (ECL plus, Amersham Pharmacia Biotech) after exposure to Hyperfilm (Amersham Pharmacia Biotech). Isolated islets fromob/ob mice were incubated for 1 h in RPMI 1640 culture medium containing 3 or 17 mm glucose and supplements as described above. The islets were washed three times in homogenization buffer containing (in mm): 20 HEPES, 1 MgCl2, 250 d-sucrose, 2 EDTA, 1 phenylmethylsulfonyl fluoride, 3 or 17 glucose as well as 5 μg/ml each of antipain, aprotinin, leupeptin, and pepstatin, pH 7.4. Thereafter the islets were homogenized in 250 μl of homogenization buffer and shortly centrifuged to pellet nuclei. The supernatant was collected and the pellet was subjected to another homogenization and centrifugation as described above. The two supernatants were pooled and loaded onto a 4.4-ml linear sucrose density gradient (prepared from 0.6 and 2m sucrose stock solutions). The gradient was centrifuged at 35,000 rpm for 18 h in a Beckman L8–55 ultracentrifuge in a SW50 rotor and 15–16 fractions (300 μl each) were collected from the top of the gradient. The linearity of the gradients was examined by measuring the refractive index of each fraction. The fractions were frozen until analyzed by immunoblotting. Equal amounts of protein from each fraction were subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were incubated with appropriate antibodies followed by ECL as described above. Chemiluminescence was detected by using a CCD camera (LAS 1000, Fuji Photo Film Co., Ltd.), that provides optimal linearity of signal intensity. The obtained signals were quantified using the Image Gauge 3.12 software (Fuji Photo Film Co., Ltd.). β-Cells isolated from ob/ob mice or lean mice were preincubated for 4 h in RPMI 1640 culture medium with additions as described above, and supplemented with 10 μm roscovitine, a potent inhibitor of Cdk5 activity (47Meijer L. Borgne A. Mulner O. Chong J.P. Blow J.J. Inagaki N. Inagaki M. Delcros J.G. Moulinoux J.P. Eur. J. Biochem. 1997; 243: 527-536Crossref PubMed Scopus (1194) Google Scholar), or with an equal volume of Me2SO as control. Insulin release was studied in perifused β-cell aggregates mixed with Bio-Gel P-4 polyacrylamide beads (Bio-Rad), in a 0.5-ml column at 37 °C. The cells were perifused with 3 mm glucose for 20 min, followed by a 20-min exposure to 11 mm glucose, returned back to 3 mm glucose for 20 min, and finally co-stimulated with 11 mm glucose and 25 mm KCl for 20 min. Roscovitine and/or Me2SO were included in all solutions during the experiment. The flow rate was 0.2 ml/min and 2-min fractions were collected. Insulin release was assayed by solid phase radioimmunoassay (RIA), using rat insulin as standard (Novo Nordisk). The basal level of insulin secretion was determined as the mean value from 10 data points of the first 20 min in 3 mm glucose. The area under the curve was examined by cutting out the curves, using the mean values, and weighing the obtained pieces on an analysis scale. Single mouse pancreatic β-cells from adult ob/ob mice were transfected with either wild-type (wt) pCMV-Cdk5 or dominant-negative (dn) pCMV-Cdk5 in combination with pIRES2-EGFP (CLONTECH), using the LipofectAMINE 2000 technique (Life Technologies, Inc.). For mock transfection, only pIRES2-EGFP was transfected. The transfected cells were cultured for 2–4 days on coverslips. The cells expressing enhanced green fluorescent protein (EGFP) were selected for whole cell patch clamp capacitance measurements. Electrodes were made from borosilicate glass capillaries, fire-polished, and coated with Sylgard close to the tips. The electrode resistance ranged between 2 and 4 MΩ when the pipettes were filled with the intracellular solutions. The electrode offset potential was corrected in the bath prior to gigaseal formation. Conventional whole cell recordings were performed with an EPC-9 patch clamp amplifier together with LockIn extension of PULSE software (HEKA Elektronik). A sinewave stimulus (500 Hz, 20 mV peak to peak) was superimposed onto a DC holding potential of −70 mV. The resulting currents were filtered at 2 kHz and sampled at 10 kHz. The capacitance traces were imported to IGOR Pro (WaveMetrics, Inc.). The data were analyzed with a PC using IGOR Pro. The standard extracellular solution consisted of (in mm): 138 NaCl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 3 d-glucose, and 5 HEPES (pH adjusted to 7.4 with NaOH). The electrode solution was composed of (in mm): 110 potassium glutamate, 10 KCl, 10 NaCl, 1 MgCl2, 10 EGTA, 7 CaCl2, 0.5 Mg-ATP, 0.1 cAMP, and 5 HEPES (pH adjusted to 7.15 with KOH). The free Ca2+concentration in the electrode solution was estimated to 0.34 μm according to the binding constants of Ca2+to EGTA and ATP in the presence of Mg2+ at pH 7.15 (48Martell A.E. Smith R.M. Vol. 1, Amino Acids, and Vol. 2, Amines. Plenum Press, New York1971Google Scholar). Cells were perfused continuously with the extracellular solution, at a flow rate of 2 ml/min during the course of an experiment. The temperature of the extracellular solution was 32 °C when measured in the position of recording electrodes. Cell aggregates attached to coverslips were loaded with 2 μmof the fluorescent Ca2+ indicator fura-2/AM at 37 °C for 45 min in a basal buffer containing (in mm): 125 NaCl, 5.9 KCl, 1.28 CaCl2, 1.2 MgCl2, 25 HEPES, 3 glucose, and 0.1% bovine serum albumin, pH 7.4. Coverslips were then mounted as the bottom of an open chamber placed on the stage of an inverted epifluorescence microscope (Zeiss, Axiovert 35M). The stage of the microscope was thermostatically controlled to maintain a mean temperature of 37 ± 1 °C in the chamber. The microscope was connected to a SPEX fluorolog-2 CM1T11I system for dual wavelength excitation fluorimetry. Various treatments during recordings were done using a perifusion system attached to the chamber (see below for the different treatments). The excitation wavelengths generated by two monochromators were directed to the cells by a dichroic mirror. The emitted light, selected by a 500–530-nm band pass filter, was directed to a CCD imaging system. The emissions at the two excitation wavelengths of 340 nm (F340) and 380 nm (F380) were used to calculate the fluorescence ratio (F340/F380), yielding relative changes in cytoplasmic free Ca2+ concentration, [Ca2+]i . Cells were pretreated with 10 μm roscovitine or Me2SO for 3.5–5 h, loaded with fura-2/AM for 45 min, and stimulated with 25 mm KCl for 2 min. Following these experiments, the cells were stimulated with glucose to verify that the recorded cells were glucose responsive β-cells. Cells from each group were averaged for each time point and a composite recording was plotted. Rate of initial response, peak delta ratio, and area under curve were analyzed. To examine if transcription of Cdk5 mRNA occurred in pancreatic islets we performed RT-PCR using Cdk5-specific primers. RNA isolated from brain served as a positive control. A fragment of identical size was amplified from islet and brain RNA. The length of the fragment was consistent with the expected length of about 380 base pairs, estimated from the nucleotide sequence (Fig.1 A). To investigate if the Cdk5 protein was expressed in pancreatic β-cells, β-cells and brain tissue were homogenized and ultracentrifuged to receive a soluble and a membrane fraction. Cdk5 immunoreactivity was detected in β-cell homogenate, both in soluble and membrane fractions. However, Cdk5 was enriched in the soluble fraction. The distribution of membrane-bound and soluble Cdk5 protein in β-cells was similar to the distribution of Cdk5 protein in brain homogenate (Fig. 1 B). To further confirm the presence and subcellular localization of Cdk5 protein in single β-cells we performed immunocytochemistry. Cdk5 exhibited a granular staining pattern, homogeneously spread in the cytoplasm. Double staining with insulin proved that Cdk5 was expressed in the β-cells and partially co-distributed with this hormone (Fig.1 C). These results clearly show that Cdk5 mRNA and protein are expressed in insulin-secreting pancreatic β-cells. A specialized feature of pancreatic β-cells is their responsiveness to glucose. To analyze if glucose stimulation of pancreatic β-cells changes the subcellular distribution of Cdk5 we performed sucrose density gradients on unstimulated and glucose-stimulated β-cells. The fractions were analyzed by Western blotting. In unstimulated β-cells Cdk5 immunoreactivity was mainly detected in low density fractions (fraction 2–5) corresponding to cytosolic fractions, but also in middle density fractions (fraction 6–10), resembling membrane-bound proteins (Fig.2 A, top, andC). The soluble protein α-tubulin served as a marker for cytosol (Fig. 2 A, middle) and syntaxin 1 was used as a plasma membrane marker (Fig. 2 A, bottom). In glucose-stimulated β-cells, Cdk5 immunoreactivity was still mainly detected in fractions 2–5, corresponding to the cytosol, but Cdk5 immunoreactivity in the plasma membrane fractions was notably diminished (Fig. 2, B, top, and C). The distribution of α-tubulin and syntaxin 1 immunoreactivity were approximately the same in glucose-stimulated β-cells (Fig.2 B, middle and bottom) and unstimulated β-cells (Fig. 2 A, middle andbottom). Fig. 2 C demonstrates a quantification of Cdk5 immunoreactivity in gradients presented as percentage of maximal signal. The sucrose density and protein concentration distribution of the gradients are shown in Fig. 2 D. These data show that glucose stimulation of pancreatic β-cells changed the subcellular localization of a population of Cdk5 protein, possibly by translocation of membrane bound Cdk5 to a soluble pool. To investigate if Cdk5 was involved in the regulation of insulin secretion, β-cells were pretreated with roscovitine for 4 h, a potent inhibitor of Cdk5 activity, or incubated with Me2SO as control. Insulin release was measured after perifusing the cells with 3 mmglucose, 11 mm glucose, and 11 mm glucose in combination with 25 mm KCl. The experiments were performed on cells isolated from ob/ob mice (Fig.3 A) and lean mice (Fig.3 B). Treatment with roscovitine decreased glucose-induced insulin secretion, both in cells from ob/ob and from lean mice compared with controls. After depolarization with KCl the reduction in insulin secretion was more pronounced. Roscovitine treatment of ob/ob cells reduced glucose-induced insulin release by ∼35% and insulin secretion induced by co-stimulation with glucose and KCl by ∼65% (Fig. 3 A). In cells isolated from lean mice the corresponding numbers were ∼50% (glucose) and ∼65% (glucose + KCl) (Fig. 3 B). The numbers were obtained by measuring the area under the curve for insulin secretion in control and roscovitine-treated cells. These data showed that roscovitine treatment prominently impaired insulin secretion from" @default.
• W2048787560 created "2016-06-24" @default.
• W2048787560 creator A5016727456 @default.
• W2048787560 creator A5017176612 @default.
• W2048787560 creator A5048630391 @default.
• W2048787560 creator A5063908911 @default.
• W2048787560 creator A5066383694 @default.
• W2048787560 creator A5082322134 @default.
• W2048787560 date "2001-09-01" @default.
• W2048787560 modified "2023-10-16" @default.
• W2048787560 title "Cyclin-dependent Kinase 5 Promotes Insulin Exocytosis" @default.
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