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Am J Physiol Endocrinol Metab 277: E862-E869, 1999;
0193-1849/99 $5.00
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Vol. 277, Issue 5, E862-E869, November 1999

Threonine phosphorylations induced by RX-871024 and insulin secretagogues in beta TC6-F7 cells

Jie An1, Genshi Zhao2, Lisa M. Churgay3, John J. Osborne1, John E. Hale3, Gerald W. Becker3, Gerald Gold1, Lawrence E. Stramm1, and Yuguang Shi1

1 Endocrine Research, 2 Infectious Disease, and 3 Research Technology and Proteins, Lilly Research Laboratories, Indianapolis, Indiana 46285


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Treatment of the pancreatic beta -cell line beta TC6-F7 with an imidazoline compound, RX-871024, KCl, or tolbutamide resulted in increased threonine phosphorylation of a 220-kDa protein (p220) concurrent with enhanced insulin secretion, which can be partially antagonized by diazoxide, an ATP-sensitive potassium (KATP) channel activator. Although phosphorylation of p220 was regulated by cytoplasmic free calcium concentration ([Ca2+]i), membrane depolarization alone was not sufficient to induce phosphorylation. Phosphorylation of p220 also was not directly mediated by protein kinase A, protein kinase C, or insulin exocytosis. Analysis of subcellular fractions indicated that p220 is a hydrophilic protein localized exclusively in the cytosol. Subsequently, p220 was purified to homogeneity, sequenced, and identified as nonmuscle myosin heavy chain-A (MHC-A). Stimulation of threonine phosphorylation of nonmuscle MHC-A by KCl treatment also resulted in increased phosphorylation of a 40-kDa protein, which was coimmunoprecipitated by antibody to MHC-A. Our results suggest that both nonmuscle MHC-A and the 40-kDa protein may play roles in regulating signal transduction, leading to insulin secretion.

imidazoline; free calcium concentration; nonmuscle myosin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN PANCREATIC beta -CELLS, signal transduction involved in insulin secretion is a complex process encompassing the integration and transmission of a number of metabolic, hormonal, and neuronal signals to the exocytotic machinery (14, 31, 33, 44). Depolarization of the plasma membrane as a result of closure of the ATP-sensitive potassium channel (KATP) leads to the opening of the voltage-dependent calcium channel (VDCC) (4) and the increase of the free cytosolic Ca2+ concentration ([Ca2+]i), which in turn triggers insulin release (4, 44). Several classes of antihyperglycemic agents, such as imidazolines and sulfonylureas, have been shown to affect [Ca2+]i. Sulfonylurea compounds, which are widely used to treat type II diabetes, stimulate insulin secretion primarily by inhibiting the KATP channels in the plasma membrane. By comparison, the molecular targets underlying imidazoline-stimulated insulin secretion have not been clearly identified. In addition to blocking of KATP channels and increasing [Ca2+]i, imidazolines also have been shown to potentiate glucose-stimulated insulin secretion in depolarized beta -cells (9, 46).

Whereas Ca2+ is an important second messenger that couples membrane depolarization to insulin secretion, the intervening steps that link [Ca2+]i elevation to insulin secretion have not been well characterized. Protein kinase A (PKA), protein kinase C (PKC), and Ca2+/calmodulin (CaM) kinase are considered as candidates that convey signals necessary for stimulus-secretion coupling (8). PKC and PKA have been suggested to play multiple roles in Ca2+-dependent and Ca2+-independent insulin secretion pathways, both by generating second messengers and by targeting the exocytotic machinery (1, 2, 22, 44). PKC has also been implicated in sulfonylurea- and imidazoline-stimulated insulin secretion by modulating Ca2+-independent pathways (11, 46).

Microtubule proteins and associated kinases also may play a role in insulin secretion (25, 32, 38). For instance, Ca2+-dependent phosphorylation of a microtubule-associated protein (MAP-2) and phosphorylation of myosin light chains were reported to be involved in insulin secretion and in release of other hormones and neurotransmitters (6, 16, 17, 24, 26-28). Whereas phosphorylation of vertebrate nonmuscle myosin heavy chain (MHC) has been demonstrated both in vivo and in vitro (12, 23, 29, 30, 35, 40, 42, 43), little is known about the regulation and functional significance of such phosphorylation. In lower eukaryotic organisms, MHC participates in diverse cellular processes including cell motility, cytokinesis, morphogenesis, and development (21, 39, 41, 45).

In this study, we identified and characterized a threonine-phosphorylated protein (p220), which was subsequently purified and identified as nonmuscle MHC-A. We also showed that stimulation of beta TC6-F7 cells with imidazoline and other insulin secretagogues resulted in increased threonine phosphorylation of nonmuscle MHC-A, which occurred distal to the calcium channel activation and proximal to exocytosis. In addition, we demonstrated a time-dependent phosphorylation of a 40-kDa protein that associated with nonmuscle MHC-A in response to KCl stimulation.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and chemical treatment. beta TC6-F7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 25 mM glucose (GIBCO-BRL), 15% heat-inactivated horse serum (Hyclone), and 2.5% fetal bovine serum (Hyclone) at 37°C with 10% CO2-90% air. Unless mentioned otherwise, 6.5 × 106 beta TC6-F7 cells (passage 50-55) were plated into 60-mm cell culture dishes. For experiments, cells were washed twice with phosphate-buffered saline (PBS) and then exposed to compounds dissolved in DMEM without glucose, because pilot experiments showed that there was no difference in p220 threonine phosphorylation when cells were treated with compounds in DMEM with or without glucose (data not shown). After treatment, cells were washed once with PBS and proteins were extracted in 200 µl of boiling lysis buffer (1% SDS, 1 mM sodium vanadate, 1 µM okadaic acid, and 10 mM Tris, pH 7.4). At least two independent experiments were done with each compound.

SDS-PAGE and Western blot analysis. Proteins from lysed cells were boiled for 5 min in lysis buffer and centrifuged to remove insoluble material. The protein concentration of the supernatant was determined using bicinchoninic acid (Pierce). A 4-20% gradient Tris-glycine polyacrylamide SDS gel (Novex) was loaded with 25 µg of total protein per well and run at 125 V for 3 h in running buffer containing 0.1% SDS, 192 mM glycine, and 25 mM Tris (pH 8.3) (Novex). Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane, and the membrane was blocked for 1 h with a TBST buffer (25 mM Tris, pH 7.5, 137 mM NaCl, 2.6 mM KCl, 0.1% Tween-20, and 0.2% casein). The blot was then incubated for 1 h with 1-2 µg/ml rabbit anti-phosphotyrosine, anti-phosphoserine, or anti-phosphothreonine antibody (Zymed) in the TBST. After three 5-min washes with the TBST buffer, the PVDF blot was incubated for 1 h with 0.3-0.5 µg/ml horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed) in TBST. After three 5-min washes with the TBST buffer, proxidase activity was detected using the enhanced chemiluminescence system (Amersham Life Science).

Insulin secretion assay. After beta TC6-F7 cells were treated with 3 ml of media containing the specified compound, 1 ml of the solution was collected and centrifuged at 10,000 rpm for 3 min. The supernatant was transferred to a new tube and stored at -20°C for insulin measurement using a scintillation proximity assay (SPA) procedure (3). Briefly, samples were diluted in SPA buffer (50 mM Na2HPO4, 150 mM NaCl, 25 mM EDTA, 0.01% thimerosal, and 0.1% BSA, pH 7.6) at a ratio of 1:75, and 50 µl of this dilution were added to the wells of a 96-well microtiter plate. Subsequent additions (in order) were 50 µl of 125I-labeled porcine insulin (NEN Life Sciences, ~10,000 counts · min-1 · well-1), 50 µl of guinea pig anti-rat insulin antibody (Linco Research), and 50 µl of protein A SPA beads (Amersham). The plate was covered with an adhesive plastic sheet, mixed briefly on a plate shaker, and incubated overnight at room temperature. Light emitted by the SPA beads as a result of bound radioactivity was detected using a Wallac 1450 Microbeta Scintillation Counter. Insulin concentrations were determined from standard curves of rat insulin using the program RIASYS (Lilly Research Laboratories). Results represent duplicate determinations from at least two independent studies and are reported as means ± SE. Data were analyzed using Student's t-test. A probability of P < 0.05 was considered statistically significant.

Purification and microsequencing of p220 from beta TC6-F7 cells. Subcellular fractionation was carried out according to Shi et al. (36). For large-scale purification of p220, beta TC6-F7 cells were treated with KCl (75 mM) for 5 min and homogenized in the hypotonic HEPES buffer (10 mM HEPES, PH 7.4, 1 mM MgCl2, 1 mM aminoethylisothiouronium bromide hydrobromide, 1 mM Na3VO4, and 1 mM phenylmethylsulfonyl difluoride). Protein lysate was enriched for high-molecular-weight proteins by a Centricon-100 filter unit (Amicon) and was subjected to HPLC using a DEAE ion exchange column (Pharmacia). The column was washed with buffer A (25 mM Tris, pH 8.2, 1 mM MgCl2, and 5% glycerol) and eluted with a linear gradient of 0-1,000 mM NaCl in buffer A. Positive fractions were identified by anti-phosphothreonine immunoblot analysis and purified by fast performance liquid chromatography (FPLC) using a hydroxyapitate (HA) column (Bio-Rad Laboratories). The column was washed with 20 mM potassium phosphate buffer (pH 7.0) and eluted with a linear gradient of 20-700 mM of potassium phosphate buffer (pH 7.0). Fractions containing p220 were pooled and concentrated using a Centricon-100 apparatus (Amicon). The concentrated sample was subjected to 4% Tris-glycine gel electrophoresis. The gel was then stained with a colloidal Coomassie G-250 (Novex), and p220 bands were isolated for microsequencing analysis at the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University.

Immunoprecipitation assay. All immunoprecipitation analyses were performed at 4°C. Briefly, KCl-stimulated beta TC6-F7 cells were washed twice with ice-cold PBS (GIBCO-BRL), homogenized in 1 ml of hypotonic HEPES buffer, and centrifuged at 800 g to eliminate nuclei. Samples containing equal amounts of protein were incubated for 90 min with the rabbit anti-human nonmuscle myosin antibody (Biomedical Technologies). The immunocomplex was added to 100 µl of a slurry of protein A-Sepharose (PAS) pretreated with washing buffer (10 mM HEPES, 150 mM NaCl, 10 mM benzamidine · HCl, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, and 0.5% Triton X-114) and incubated for an additional 90 min. After four brief washes, the immunocomplex was eluted from PAS by boiling in 20 µl of 2× Tris · glycine SDS sample buffer (Novex), followed by SDS-PAGE on a 4-20% Tris-glycine gradient SDS gel (Novex) and Western blot analysis.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Time- and dose-dependent phosphorylation of p220 induced by imidazoline and other depolarization reagents. To identify signal molecules involved in imidazoline-stimulated insulin secretion, we investigated protein phosphorylation in beta TC6-F7 cells treated with an insulin secretagogue, RX-871024, which contains an imidazoline ring. Protein lysates were separated by SDS-PAGE, and phosphorylated proteins were analyzed by immunoblot assays by use of antibodies against phosphoamino acids. Treatment of beta TC6-F7 cells with 100 µM RX-871024 resulted in increased threonine phosphorylation of a protein with an apparent molecular mass of 220 kDa (p220, Fig. 1A). Western blot analysis with anti-phosphotyrosine antibody also detected increases in tyrosine phosphorylation of a 42-kDa protein, which was later identified as extracellular signal-regulated mitogen-activated protein kinase, or ERK2 MAP kinase (data not shown). In contrast, initial results indicated that antibody against phosphoserine did not detect any changes in serine phosphorylation. Thus we chose to study p220 phosphorylation in greater detail because p220 was abundant and its phosphorylation was markedly increased by exposure of cells to RX-871024.


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  		Fig. 1.   Threonine phosphorylation of p220 induced by RX-871024, KCl, and glibenclamide in beta TC6-F7 cells. Cells were treated with 100 µM RX-871024 (A), 75 mM KCl (B), and 5 µM glibenclamide (C) for periods of 0, 1, 5, 10, and 20 min in Dulbecco's modified Eagle's medium. At the end of each incubation period, cells were harvested, and cell lysates were quantified using the bicinchoninic acid method. Protein lysate (25 µg) from each treatment was analyzed by SDS-PAGE, followed by Western blot analysis with anti-phosphothreonine antibody.

One of the reported mechanisms of RX-871024 on insulin secretion involved closure of ATP-regulated K+ channels and membrane depolarization (10). We therefore tested the effects of two other membrane depolarization reagents, KCl and glibenclamide. Treatment of beta TC6-F7 cells with 75 mM KCl or 5 µM glibenclamide caused time-dependent increases of p220 phosphorylation (Fig. 1, B and C). However, there were significant differences in the time course of phosphorylation of p220 for KCl vs. RX-871024 and glibenclamide. Threonine phosphorylation of p220 induced by KCl occurred instantaneously on addition of KCl and peaked in 5 min after the treatment. In comparison, p220 phosphorylation induced by RX-871024 (compare Fig. 1, A with B) or glibenclamide (compare Fig. 1, B with C) was slower in onset and peaked at 20 min.

p220 Phosphorylation and insulin secretion. RX-871024 has been reported to potentiate glucose-stimulated insulin secretion (46). However, we and others have experienced a low threshold of glucose-stimulated insulin secretion from beta TC6-F7 around 2 mM of glucose. In agreement with the lack of glucose responsiveness in beta TC6-F7 cells, p220 phosphorylation was not affected by changing levels of glucose in the media (data not shown). To investigate whether p220 threonine phosphorylation was related to insulin secretion, we analyzed in parallel insulin secretion from beta TC6-F7 cells treated with KCl and RX-871024. As shown in Fig. 2A, stimulation of beta TC6-F7 cells with 15 mM, 45 mM, or 75 mM KCl caused dose-dependent increases in insulin secretion concurrently with increased threonine phosphorylation of p220 as measured 5 min after the treatment. Levels of insulin secretion were significantly (P < 0.05) higher after treatment with 45 mM and 75 mM KCl vs. the control. Similar results were observed when Earle's balanced salt solution buffer was adjusted for osmolarity by reducing the NaCl concentration to exclude possible osmotic effects on insulin secretion (data not shown). p220 Phosphorylation was also correlated with insulin secretion from beta TC6-F7 cells treated with RX-871024. As demonstrated in Fig. 2B, 100 µM RX-871024 increased both insulin secretion and p220 threonine phosphorylation measured after 20 min of treatment compared with the negative control.


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  		Fig. 2.   Correlation between insulin secretion and p220 phosphorylation. A: dose-dependent effect of KCl on both insulin release and threonine phosphorylation of p220 in beta TC6-F7 cells. Cells were treated with KCl at concentrations of 0, 15, 45, and 75 mM, respectively, for 5 min in Earle's balanced salt solution (EBSS) buffer. B: effect of RX-871024 on insulin release and threonine phosphorylation of p220 in beta TC6-F7 cells. Cells were treated with RX-871024 (100 µM) for 0, 5, and 20 min in EBSS buffer. The presence and absence of the compound was shown as "+" and "-", respectively. At the end of each incubation period, the supernatant was removed for insulin quantification using a scintillation proximity assay. * Significantly different from negative control (P < 0.05).

p220 Phosphorylation distal to Ca2+ channel activation. The effect of membrane potential on p220 phosphorylation was further investigated by treatment of beta TC6-F7 cells with diazoxide, a KATP channel activator. As indicated by Fig 3A, both p220 phosphorylation and insulin secretion were inhibited by the presence of 150 µM diazoxide in the media. Because membrane depolarization of beta -cells often leads to activation of Ca2+ channels, we next investigated whether Ca2+ channels played a role in regulating p220 phosphorylation. As shown in Fig. 3B, pretreatment of beta TC6-F7 cells with 50 µM nifedipine, an L-type Ca2+ channel blocker, completely abolished both insulin secretion and p220 phosphorylation induced by treatment of 75 mM KCl. In the absence of KCl, nifedipine did not have any effect, either on p220 phosphorylation or on insulin secretion (Fig. 3B).


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  		Fig. 3.   Effects of diazoxide, nifedipine, and extracellular Ca2+ on KCl-induced insulin release and threonine phosphorylation of p220 in beta TC6-F7 cells. A: cells were pretreated with diazoxide (150 µM) for 5 min and then treated with KCl (75 mM) for 5 min in EBSS buffer containing 10 mM glucose. B: cells were pretreated with or without nifedipine (50 µM) for 15 min and then treated with or without KCl (75 mM) for 5 min in EBSS buffer. C: cells were treated with or without KCl (75 mM) for 5 min in EBSS buffer containing glucose (10 mM) in the presence or absence of calcium (1.8 mM). At the end of each treatment period, the supernatant was removed for insulin quantification. The presence or absence of a compound was shown as "+" and "-", respectively. * Significantly different from negative controls (P < 0.05).

RX-871024 stimulates insulin release by both Ca2+-dependent and Ca2+-independent mechanisms (46). To separate the effect of membrane depolarization from the effect of [Ca2+]i on p220 phosphorylation, we next compared p220 phosphorylation in beta TC6-F7 cells cultured in media with and without Ca2+. As indicated by Fig. 3C, treatment of beta TC6-F7 cells with 75 mM KCl induced strong p220 phosphorylation concurrent with insulin secretion only in the presence of 1.8 mM extracellular Ca2+, suggesting that membrane depolarization alone was not sufficient to induce p220 phosphorylation. Extracellular Ca2+ alone in the absence of depolarizing concentration of KCl did not stimulate p220 phosphorylation (Fig. 3C).

Regulation of p220 phosphorylation by PKA and PKC. PKA has been implicated to play a role in insulin secretion stimulated by RX-871024 (46). To further understand the signal events that regulate p220 threonine phosphorylation, we tested the effect of IBMX on p220 phosphorylation. IBMX, an inhibitor of phosphodiesterase, potentiates glucose-induced insulin secretion by raising cAMP levels and activating the PKA-mediated signal transduction pathway (15). Whereas neither glucose nor IBMX alone stimulated insulin secretion from the cells, treatment of beta TC6-F7 cells with IBMX (100 µM) in the presence of glucose (10 mM) resulted in a sharp increase in insulin secretion as measured at 5 and 20 min after the stimulation (Fig. 4). In contrast to the treatment by KCl, no p220 phosphorylation was induced by insulin secretion stimulated by IBMX either at 5 or 20 min after the treatment. Thus p220 phosphorylation is not directly regulated by PKA. The results also suggest that p220 phosphorylation is not a signal event obligatory to insulin exocytosis. Likewise, p220 phosphorylation was not affected by treatment of beta TC6-F7 cells with phorbol 12-myristate 13-acetate (PMA), an activator of PKC, and H7, an inhibitor of PKC, suggesting that p220 is not a substrate of PKC (data not shown).


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  		Fig. 4.   Comparison of effects of IBMX and KCl on insulin release and threonine phosphorylation of p220 in beta TC6-F7 cells. Cells were treated with IBMX (100 µM) or KCl (75 mM) in the presence or absence of glucose (10 mM) for 0, 5, and 20 min. At the end of the treatment period, the supernatant was removed for insulin quantification. Cells were harvested, and cell lysates were analyzed as described in Fig. 1. The presence or absence of a compound was shown as "+" and "-", respectively. * and + Significantly different from negative controls (P < 0.05); different symbols represent different control groups.

Phosphorylated p220 is a cytosolic protein. Subcellular fractionation was carried out to characterize the biochemical features of p220 and to facilitate purification of p220 from beta TC6-F7 cells. After treatment with 75 mM KCl for 1 and 5 min to stimulate p220 phosphorylation, beta TC6-F7 cells were homogenized and separated into a cytosol (C, Fig. 5) and a particulate (P, Fig. 5) membrane fraction. The membrane fraction was washed in 0.5 M NaCl. Triton X-114 extracts of cytosol and membrane fractions were subjected to temperature-induced Triton X-114 phase transition. The aqueous phase (A, Fig. 5), which contained hydrophilic proteins, and the detergent phase (D, Fig. 5), which contained hydrophobic proteins, were separated by SDS-PAGE and subjected to immunoblotting analysis using anti-phosphothreonine antibodies. As indicated by Fig. 5, phosphorylated p220 was a hydrophilic protein and localized exclusively in the aqueous phase of the cytosolic fraction.


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  		Fig. 5.   Subcellular localization of p220 in beta TC6-F7 cells. Cells were treated with 75 mM of KCl for 1 (lanes 1-4) and 5 min (lanes 5-8) before subcellular fractionation. A cytosolic fraction (C) and an extract of a washed particulate fraction (P) were prepared from beta TC6-F7 cells and subjected to temperature-induced Triton X-114 phase separation. Aqueous (A) and detergent (D) phases were analyzed by SDS-PAGE immunoblot analysis with anti-phosphothreonine antibody. Positions of molecular mass markers (M) are indicated with numbers on left. The position of p220 is indicated with an arrow.

Purification of p220 and identification as nonmuscle MHC. p220 was purified from beta TC6-F7 cells treated with 75 mM KCl. Cytosolic fractions enriched for high molecular proteins by Centricon filtration were separated by ion exchange chromatography by use of a DEAE column. Column fractions containing phosphorylated p220 were identified by Western blot analysis using anti-phosphothreonine antibodies. Combined fractions containing p220 were further purified on an HA column, which resulted in significant enrichment of p220, as indicated in Fig. 6. As a final purification step, p220 was separated from other proteins by SDS-PAGE on 4% Tris-glycine gels. After Coomassie blue staining of the gel, a p220 band was isolated and subjected to microsequencing analysis.


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  		Fig. 6.   Immunoblot analysis of purified fractions from a hydroxyapitate (HA)-fast performance liquid chromatography (FPLC) column. Column fractions were separated by 4-20% SDS-PAGE followed by Western blot analysis using anti-phosphothreonine antibody. Lane 1, a fraction containing p220 from a DEAE-HPLC column. Lane M, molecular mass marker. Lanes 7-10, positive fractions containing p220 from HA-FPLC column purification. Position of p220 is indicated with an arrow.

Two peptide sequences were generated from the microsequencing analysis. The first peptide contained the sequence R-G-D-L-P-F-V-V-T-R, and the second contained the sequence T-D-L-L-L-E-P-Y-N-K. Each peptide sequence was compared with proteins in the SwissProt database. Both peptides shared high sequence homology with vertebrate nonmuscle MHC-A. The first peptide shares 100% homology with a sequence fragment located at the COOH terminus of chick nonmuscle MHC-A (Fig. 7) and matched all but one amino acid of a sequence located in the corresponding region of the human nonmuscle MHC-A (Fig. 7). The second peptide shared 100% sequence identity with a peptide sequence close to the NH2 terminus of both human and chick nonmuscle MHC-A (Fig. 7). No matches were found with any other members of the myosin super family, including nonmuscle MHC-B, which shares high homology with nonmuscle MHC-A, or any other protein sequences in the data bank. This was also supported by immunoprecipitation and immunoblot analysis by use of a polyclonal nonmuscle MHC antibody and partially purified DEAE fractions. Only p220-positive fractions contained proteins that were immunoreactive with nonmuscle MHC antibody (data not shown), suggesting that the threonine-phosphorylated p220 is the nonmuscle MHC-A.


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  		Fig. 7.   Sequence comparison of p220 peptides with nonmuscle myosin heavy chain A (MHC-A) from human and chick. Nos. indicate position of peptide in myosin protein sequence. The only mismatch of p220 peptide sequence with human nonmuscle MHC-A is underlined.

Immunoprecipitation analyses were carried out to verify whether nonmuscle MHC-A becomes phosphorylated on threonine residue(s) in response to insulin secretion by use of human nonmuscle MHC-A antibody and lysates from beta TC6-F7 cells treated with KCl (Fig. 8). The immunoprecipitates were characterized by immunoblot analysis with anti-phosphothreonine antibody (Fig. 8A) and nonmuscle MHC antibody (Fig 8B). Treatment of beta TC6-F7 with 75 mM KCl resulted in time-dependent threonine phosphorylation of nonmuscle MHC identical to that of p220 (compare Fig. 8, A with B). As indicated by Fig. 8B, roughly equal amounts of nonmuscle MHC-A were immunoprecipitated from the lysates of beta TC6-F7 cells treated with KCl for different lengths of time. In addition to nonmuscle MHC phosphorylation, a protein with molecular mass of ~40 kDa (p40) was coimmunoprecipitated by the nonmuscle MHC antibody. The p40 protein also demonstrated time-dependent phosphorylation on threonine residue(s) in response to the KCl treatment (Fig. 8A). The p40 protein was not recognized by the nonmuscle MHC antibody from the Western blot analysis (Fig. 8B), suggesting that the molecule is not a degraded product of p220.


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  		Fig. 8.   Nonmuscle MHC threonine phosphorylation and association with the p40 protein induced by KCl in beta TC6-F7 cells. Cells were treated with 75 mM KCl for 0 (lane 1), 1 (lane 2), 5 (lane 3), 10 (lane 4), and 20 min (lane 5), respectively. Immunoprecipitation was carried out using human nonmuscle MHC antibody, followed by Western blot analyses using rabbit anti-phosphothreonine antibody (A) and rabbit anti-nonmuscle MHC antibody (B). Positions of molecular mass markers are indicated with nos. on left. Positions of nonmuscle MHC and p40 proteins are indicated with arrows.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The signal molecules involved in the insulinotropic effects of RX-871024 have not been well characterized. In the present study, we have demonstrated for the first time that stimulation of pancreatic beta -cells with RX-871024 leads to increased threonine phosphorylation of a 220-kDa protein (p220). Whereas imidazolines have been shown to potentiate glucose-stimulated insulin secretion (46), our data showed that p220 phosphorylation stimulated by RX-871024 was mainly related to the membrane depolarization effect of imidazolines. This is supported by our experiments showing that p220 phosphorylation also occurs in beta -cells treated with KCl and glibenclamide. Although these three secretagogues differ in mechanisms involved in insulin secretion, all have been shown to cause beta -cell depolarization, activation of L-type VDCC, and increase in [Ca2+]i. Indeed, our data indicate that p220 phosphorylation can be blocked by treatment with diazoxide, a KATP channel activator, by treatment with nifedipine, an L-type Ca2+-channel inhibitor, and by depletion of extracellular calcium. Our data also suggest that depolarization without affecting [Ca2+]i was not sufficient to elicit p220 phosphorylation.

Through protein purification and peptide sequencing analyses, we identified p220 as nonmuscle MHC-A. Subsequent immunoprecipitation analyses also confirmed that the phosphorylation profile of nonmuscle MHC coincided with that of p220 when cells were tested with KCl. Two isoforms of MHC, MHC-A and MHC-B, were reported to be present in mammals (18, 19, 34, 37). Even though both forms were recently found to be expressed in pancreatic beta -cells (43), our sequence data suggest that the A form is the predominant one that is responsive to stimulation of acute insulin secretion. This is also consistent with a recent report (16) that only a single form of MHC was detected in pancreatic beta -cells by immunoblot analysis. However, it is possible that the B isoform was also responsive to KCl stimulation but was not detected without enrichment of the protein by immunoprecipitation, because our immunoprecipitation analysis using nonmuscle MHC antibody resulted in detection of two isoforms (Fig. 8). Interestingly, threonine phosphorylation of both forms appeared to be equally stimulated by KCl treatment, as demonstrated by immunoprecipitation analysis.

One of the suggested mechanisms of RX-871024 involves a direct stimulation of insulin exocytosis (9). A potential role of MHC phosphorylation in stimulating exocytosis was tested in our experiments by treatment of beta -cells with nondepolarizing secretagogues, such as IBMX. It was expected that if MHC phosphorylation were an obligatory event that led to insulin exocytosis, p220 phosphorylation should also occur in exocytosis stimulated by different mechanisms. We chose to use IBMX because the secretagogue stimulates insulin secretion and bypasses the membrane depolarization step. IBMX potentiates glucose-stimulated insulin secretion by increasing the level of cAMP, which activates PKA-mediated signal transduction pathways in beta -cells. Whereas IBMX elicited a sharp increase in insulin release in the presence of high glucose, p220 phosphorylation was not affected by IBMX. The results suggest that phosphorylation of nonmuscle MHC is not an obligatory event in insulin exocytosis. Our data also suggest that MHC phosphorylation is not mediated by PKA.

A number of kinases, including PKC, CaM kinase, and casein kinase II, have been suggested to phosphorylate MHC both in lower eukaryotic systems and in mammalian cells (5, 20, 40). However, the only kinase with demonstrated specificity for MHC came from biochemical and molecular characterization of Dictyostelium myosin heavy chain kinase, or MHCK (13). Because PKC has been implicated in insulin release stimulated by imidazoline and glibenclamide (11, 46), we also tested the involvement of PKC in regulating MHC phosphorylation by using an inhibitor and an activator of the kinase. Our data showed that p220 phosphorylation was not affected by either the PKC activator PMA or the PKC inhibitor H7. The results suggest that MHC threonine phosphorylation is not directly regulated by the PKC-mediated signal transduction pathways, a conclusion which is consistent with previous reports that the PKC-mediated phosphorylation of MHC primarily affects serine residues (7, 40).

One of the interesting observations from our current studies involved identification of a 40-kDa (p40) molecule associated with p220 phosphorylation. Because p40 was identified only after immunoprecipitation, it is possible that the protein was nonspecifically immunoprecipitated by the polyclonal MHC antibody. Alternatively, p40 may be physically associated with MHC and hence enriched by the immunoprecipitation of nonmuscle MHC. This was partly supported by the observation that p40 was the only other threonine-phosphorylated protein immunoprecipitated by the MHC antibody among all the threonine-phosphorylated proteins in the cell lysate, and that p40 protein was not recognized by the polyclonal MHC antibody on Western blot after enrichment with immunoprecipitation. Furthermore, p40 was also responsive to KCl stimulation and demonstrated time-dependent phosphorylation on threonine residue(s). Interestingly, the threonine phosphorylation of p40 appeared to be at a different rate than the phosphorylation of nonmuscle MHC. In comparison with the threonine phosphorylation of nonmuscle MHC, which peaked at 5 min after KCl treatment, p40 phosphorylation became more pronounced after 10 min of treatment. The molecular identity of p40 remains unknown, and its identification may help explain how nonmuscle MHC phosphorylation is regulated in pancreatic beta -cells. More importantly, the identification of p40 also may reveal a missing link between nonmuscle MHC phosphorylation and signal transduction pathways involved in acute insulin secretion.


    ACKNOWLEDGEMENTS

We thank Valerie X. Williams, Carol Broderick, and Dr. Xiaodong Liu for technical assistance, and Drs. Christopher Newgard, Mark L. Heiman, Michael Statnick, and Scott E. Hayes for critically reading the manuscript.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for correspondence and reprint requests: Y. Shi, Endocrine Division, DC 0545, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285 (E-mail: Shi_Yuguang{at}Lilly.com).

Received 19 February 1999; accepted in final form 11 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ammala, C., L. Eliasson, K. Bokvist, P. O. Berggren, R. E. Honkanen, A. Sjoholm, and P. Rorsman. Activation of protein kinases and inhibition of protein phosphatases play a central role in the regulation of exocytosis in mouse pancreatic beta cells. Proc. Natl. Acad. Sci. USA 91: 4343-4347, 1994[Abstract/Free Full Text].

2.   Basudev, H., P. M. Jones, and S. L. Howell. Protein phosphorylation in the regulation of insulin secretion: the use of site-directed inhibitory peptides in electrically permeabilised islets of Langerhans. Acta Diabetol. 32: 32-37, 1995[Medline].

3.   Bosworth, N., and P. Towers. Scintillation proximity assay. Nature 341: 167-168, 1989[Medline].

4.   Boyd, A. E., III. The role of ion channels in insulin secretion. J. Cell. Biochem. 48: 235-241, 1992[Medline].

5.   Brzeska, H., and E. D. Korn. Regulation of class I and class II myosins by heavy chain phosphorylation. J. Biol. Chem. 271: 16983-16986, 1996[Free Full Text].

6.   Choi, O. H., R. S. Adelstein, and M. A. Beaven. Secretion from rat basophilic RBL-2H3 cells is associated with diphosphorylation of myosin light chains by myosin light chain kinase as well as phosphorylation by protein kinase C. J. Biol. Chem. 269: 536-541, 1994[Abstract/Free Full Text].

7.   Conti, M. A., J. R. Sellers, R. S. Adelstein, and M. Elzinga. Identification of the serine residue phosphorylated by protein kinase C in vertebrate nonmuscle myosin heavy chains. Biochemistry 30: 966-970, 1991[Medline].

8.   Easom, R. A., N. R. Filler, E. M. Ings, J. Tarpley, and M. Landt. Correlation of the activation of Ca2+/calmodulin-dependent protein kinase II with the initiation of insulin secretion from perifused pancreatic islets. Endocrinology 138: 2359-2364, 1997[Abstract/Free Full Text].

9.   Efanov, A. M., S. V. Zaitsev, I. B. Efanova, S. Zhu, C.-G. Östenson, P.-O. Berggren, and S. Efendic. Signaling and sites of interaction for RX-871024 and sulfonylurea in the stimulation of insulin release. Am. J. Physiol. 274 (Endocrinol. Metab. 37): E751-E757, 1998[Abstract/Free Full Text].

10.   Efanova, I. B., S. V. Zaitsev, G. Brown, P. O. Berggren, and S. Efendic. RX871024 induces Ca2+ mobilization from thapsigargin-sensitive stores in mouse pancreatic beta-cells. Diabetes 47: 211-218, 1998[Abstract].

11.   Eliasson, L., E. Renstrom, C. Ammala, P. O. Berggren, A. M. Bertorello, K. Bokvist, A. Chibalin, J. T. Deeney, P. R. Flatt, J. Gabel, J. Gromada, O. Larsson, P. Lindstrom, C. J. Rhodes, and P. Rorsman. PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells. Science 271: 813-815, 1996[Abstract].

12.   Fechheimer, M., and J. J. Cebra. Phosphorylation of lymphocyte myosin catalyzed in vitro and in intact cells. J. Cell Biol. 93: 261-268, 1982[Abstract/Free Full Text].

13.   Futey, L. M., Q. G. Medley, G. P. Cote, and T. T. Egelhoff. Structural analysis of myosin heavy chain kinase A from Dictyostelium. Evidence for a highly divergent protein kinase domain, an amino-terminal coiled-coil domain, and a domain homologous to the beta-subunit of heterotrimeric G proteins. J. Biol. Chem. 270: 523-529, 1995[Abstract/Free Full Text].

14.   Henquin, J. C., M. Gembal, P. Detimary, Z. Y. Gao, C. Warnotte, and P. Gilon. Multisite control of insulin release by glucose. Diabete Metab. 20: 132-137, 1994[Medline].

15.   Hill, R. S., J. M. Oberwetter, and A. E. Boyd III. Increase in cAMP levels in beta-cell line potentiates insulin secretion without altering cytosolic free-calcium concentration. Diabetes 36: 440-446, 1987[Abstract].

16.   Iida, Y., T. Senda, Y. Matsukawa, K. Onoda, J.-I. Miyazaki, H. Sakaguchi, Y. Nimura, H. Hidaka, and I. Niki. Myosin light-chain phosphorylation controls insulin secretion at a proximal step in the secretory cascade. Am. J. Physiol. 273 (Endocrinol. Metab. 36): E782-E789, 1997[Abstract/Free Full Text].

17.   Jefferson, A. B., S. M. Travis, and H. Schulman. Activation of multifunctional Ca2+/calmodulin-dependent protein kinase in GH3 cells. J. Biol. Chem. 266: 1484-1490, 1991[Abstract/Free Full Text].

18.   Katsuragawa, Y., M. Yanagisawa, A. Inoue, and T. Masaki. Two distinct nonmuscle myosin-heavy-chain mRNAs are differentially expressed in various chicken tissues. Identification of a novel gene family of vertebrate non-sarcomeric myosin heavy chains. Eur. J. Biochem. 184: 611-616, 1989[Medline].

19.   Kawamoto, S., and R. S. Adelstein. Chicken nonmuscle myosin heavy chains: differential expression of two mRNAs and evidence for two different polypeptides. J. Cell Biol. 112: 915-924, 1991[Abstract/Free Full Text].

20.   Kelley, C. A., S. Kawamoto, M. A. Conti, and R. S. Adelstein. Phosphorylation of vertebrate smooth muscle and nonmuscle myosin heavy chains in vitro and in intact cells. J. Cell. Sci., Suppl. 14: 49-54, 1991[Medline].

21.   Kelley, C. A., F. Oberman, J. K. Yisraeli, and R. S. Adelstein. A Xenopus nonmuscle myosin heavy chain isoform is phosphorylated by cyclin-p34cdc2 kinase during meiosis. J. Biol. Chem. 270: 1395-1401, 1995[Abstract/Free Full Text].

22.   Kindmark, H., M. Koohler, O. Larsson, A. Khan, and P. O. Berggren. Dissociation between exocytosis and Ca2+-channel activity in mouse pancreatic beta-cells stimulated with calmidazolium (compound R24571). FEBS Lett. 369: 315-320, 1995[Medline].

23.   Korn, E. D., and J. A. Hammer III. Myosins of nonmuscle cells. Annu. Rev. Biophys. Biophys. Chem. 17: 23-45, 1988[Medline].

24.   Krueger, K. A., H. Bhatt, M. Landt, and R. A. Easom. Calcium-stimulated phosphorylation of MAP-2 in pancreatic beta TC3-cells is mediated by Ca2+/calmodulin-dependent kinase II. J. Biol. Chem. 272: 27464-27469, 1997[Abstract/Free Full Text].

25.   Lacy, P. E., M. M. Walker, and C. J. Fink. Perifusion of isolated rat islets in vitro. Participation of the microtubular system in the biphasic release of insulin. Diabetes 21: 987-998, 1972[Medline].

26.   Ludowyke, R. I., I. Peleg, M. A. Beaven, and R. S. Adelstein. Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells. J. Biol. Chem. 264: 12492-12501, 1989[Abstract/Free Full Text].

27.   Ludowyke, R. I., L. L. Scurr, and C. M. McNally. Calcium ionophore-induced secretion from mast cells correlates with myosin light chain phosphorylation by protein kinase C. J. Immunol. 157: 5130-5138, 1996[Abstract].

28.   MacDonald, M. J., and A. Kowluru. Calcium-calmodulin-dependent myosin phosphorylation by pancreatic islets. Diabetes 31: 566-570, 1982[Abstract].

29.  Moussavi, R. S., C. A. Kelley, and R. S. Adelstein. Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains. Mol. Cell. Biochem. 127-128 and 219-227, 1993.

30.   Muhlrad, A., and A. Oplatka. Phosphorylation of fibroblast myosin. FEBS Lett. 77: 37-40, 1977[Medline].

31.   Newgard, C. B., and J. D. McGarry. Metabolic coupling factors in pancreatic beta-cell signal transduction. Annu. Rev. Biochem. 64: 689-719, 1995[Medline].

32.   Pipeleers, D. G., M. A. Pipeleers-Marichal, and D. M. Kipnis. Microtubule assembly and the intracellular transport of secretory granules in pancreatic islets. Science 191: 88-90, 1976[Abstract/Free Full Text].

33.   Prentki, M., K. Tornheim, and B. E. Corkey. Signal transduction mechanisms in nutrient-induced insulin secretion. Diabetologia 40, Suppl.: S32-S41, 1997.

34.   Saez, C. G., J. C. Myers, T. B. Shows, and L. A. Leinwand. Human nonmuscle myosin heavy chain mRNA: generation of diversity through alternative polyadenylylation. Proc. Natl. Acad. Sci. USA 87: 1164-1168, 1990[Abstract/Free Full Text].

35.   Sagara, J., K. Nagata, and Y. Ichikawa. Phosphorylation of the myosin heavy chain. Its effect on actin-activated Mg2+-stimulated ATPase in leukaemic myeloblasts. Biochem. J. 214: 839-843, 1983[Medline].

36.   Shi, Y., B. Veit, and S. Baekkeskov. Amino acid residues 24-31 but not palmitoylation of cysteines 30 and 45 are required for membrane anchoring of glutamic acid decarboxylase, GAD65. J. Cell Biol. 124: 927-934, 1994[Abstract/Free Full Text].

37.   Simons, M., M. Wang, O. W. McBride, S. Kawamoto, K. Yamakawa, D. Gdula, R. S. Adelstein, and L. Weir. Human nonmuscle myosin heavy chains are encoded by two genes located on different chromosomes. Circ. Res. 69: 530-539, 1991[Abstract/Free Full Text].

38.   Somers, G., B. Blondel, L. Orci, and W. J. Malaisse. Motile events in pancreatic endocrine cells. Endocrinology 104: 255-264, 1979[Abstract].

39.   Spudich, J. A. In pursuit of myosin function. Cell Regul. 1: 1-11, 1989[Medline].

40.   Tan, J. L., S. Ravid, and J. A. Spudich. Control of nonmuscle myosins by phosphorylation. Annu. Rev. Biochem. 61: 721-759, 1992[Medline].

41.   Tullio, A. N., D. Accili, V. J. Ferrans, Z. X. Yu, K. Takeda, A. Grinberg, H. Westphal, Y. A. Preston, and R. S. Adelstein. Nonmuscle myosin II-B is required for normal development of the mouse heart. Proc. Natl. Acad. Sci. USA 94: 12407-12412, 1997[Abstract/Free Full Text].

42.   Watanabe, T. K., E. R. Kuczmarski, and J. K. Reddy. Myosin from pancreatic acinar carcinoma cells. Isolation, characterization and demonstration of heavy- and light-chain phosphorylation. Biochem. J. 247: 513-518, 1987[Medline].

43.   Wilson, J. R., R. I. Ludowyke, and T. J. Biden. Nutrient stimulation results in a rapid Ca2+-dependent threonine phosphorylation of myosin heavy chain in rat pancreatic islets and RINm5F cells. J. Biol. Chem. 273: 22729-22737, 1998[Abstract/Free Full Text].

44.   Wollheim, C., J. Lang, and R. Regazzi. The exocytotic process of insulin secretion and its regulation by Ca2+ and G-proteins. Diabetes Rev. 4: 276-297, 1996.

45.   Young, P. E., A. M. Richman, A. S. Ketchum, and D. P. Kiehart. Morphogenesis in Drosophila requires nonmuscle myosin heavy chain function. Genes Dev. 7: 29-41, 1993[Abstract/Free Full Text].

46.   Zaitsev, S. V., A. M. Efanov, I. B. Efanova, O. Larsson, C. G. Ostenson, G. Gold, P. O. Berggren, and S. Efendic. Imidazoline compounds stimulate insulin release by inhibition of KATP channels and interaction with the exocytotic machinery. Diabetes 45: 1610-1618, 1996[Abstract].

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