Phosphorylation of F-actin-Associating G Protein gamma 12 Subunit Enhances Fibroblast Motility*

Hiroshi UedaDagger , Junji Yamauchi§, Hiroshi Itoh§, Rika MorishitaDagger , Yoshito Kaziro§, Kanefusa KatoDagger , and Tomiko AsanoDagger

From the Dagger  Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392 and the § Department of Biological Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan

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Eleven isoforms of G protein gamma  subunit have been found thus far, but the precise roles of individual gamma  subunits are not known. The gamma 12 subunit has two unique properties: phosphorylation by protein kinase C and association with F-actin. To elucidate the role of gamma 12, we overexpressed gamma 12 and other gamma  subunits in NIH 3T3 cells together with the beta 1 subunit. The overexpressed gamma 12 as well as endogenous gamma 12, but not gamma 2, gamma 5, and gamma 7 subunits, associated with cytoskeletal components. Expression of gamma 12 induced remarkable changes including cell rounding, disruption of stress fibers, and enhancement of cell migration, but expression of other gamma  subunits did not induce significant changes. Deletion of the N-terminal region of gamma 12 decreased the abilities of gamma 12 to associate with cytoskeletal fractions, to induce cell rounding, and to increase cell motility. Replacement by alanine of Ser2 of gamma 12 (Ser1 of a mature gamma 12 protein), a phosphorylation site for protein kinase C, eliminated these effects of gamma 12, whereas a mutant in which Ser2 was replaced with glutamic acid showed effects equivalent to wild-type gamma 12. These results indicate that phosphorylation of gamma 12 at Ser2 enhances the motility of cells.

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Heterotrimeric G proteins1 play a major role in signal transduction from cell surface receptors to intracellular effectors. The beta gamma complexes, as well as alpha  subunits, directly regulate various effectors, including adenylyl cyclase, phospholipase C-beta , phosphatidylinositol 3-kinase, K+ channels, and Ca2+ channels (1, 2). At present, 11 isoforms of the gamma  subunit have been found (2-6). Although the biological properties of the beta gamma complex containing gamma 1 are noticeably different from those of beta gamma complexes containing the other gamma  subunits (7, 8), the precise roles of individual gamma  subunits are not known.

The gamma 12 subunit, which is widely distributed and especially rich in fibroblasts and smooth muscle cells (6), has unique properties. First, gamma 12 is a selective substrate for protein kinase C (PKC) (6, 9). In Swiss 3T3 cells, gamma 12 is phosphorylated upon exposure of cells to various reagents such as phorbol 12-myristate 13-acetate, serum, lysophosphatidic acid, endothelines, and growth factors (9). Phosphorylated gamma 12 enhanced the association of beta gamma 12 with Goalpha (6) and weakened the ability of beta gamma 12 to stimulate type II adenylyl cyclase (10), but the magnitudes of the changes induced by phosphorylation were relatively small. The second unique property is that gamma 12 associates with F-actin in cells and in a cell-free system (11). In addition to gamma 12, various alpha  subunits, such as Gi2alpha , Gsalpha (12), and Gq/11alpha (13), and the beta  subunit (14) as well as enzymes involved in signal transduction, such as phospholipase C, phosphoinositide 3-kinase, and PKC (15-17) are found associated with the cytoskeleton in a variety of cells, but the physiological significance of these associations is unclear.

To examine the role of gamma 12, we overexpressed gamma 12, other gamma  subunits and their mutants together with beta 1 in NIH 3T3 cells. The results indicate that only gamma 12 subunit induces cell rounding and enhances cell migration and that phosphorylation of gamma 12 is involved in these processes.

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Plasmids and Antibodies-- cDNAs of several gamma 12 and gamma 2 mutants were prepared using synthetic polymerase chain reaction primers (3, 6). cDNAs of bovine beta 1 (18) and gamma 2 (3) were generously provided by Dr. M. I. Simon (California Institute of Technology) and Dr. T. Nukada (Tokyo Institute of Psychiatry), respectively. All cDNAs of G protein subunits (3-6) and a C-terminal fragment (amino acids 495-689) of the beta -adrenergic receptor kinase (beta ARKct) (19) were subcloned into pCMV5 vector as described previously (20). Antibodies against the beta  and gamma  subunits of G protein have been described previously (6, 9, 21, 22).

Transfection and Staining-- NIH 3T3 cells were grown in Dulbecco's modified essential medium (DMEM) supplemented with 10% calf serum. Transfection was performed using LipofectAMINE Plus reagent (Life Technologies, Inc.) as described. After 48 h, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline and immunostained using antibodies against gamma  or beta , followed by fluorescein isothiocyanate-labeled secondary antibody (MBL) as described (11). The cells were also stained for F-actin with tetramethylrhodamine isothiocyanate-phalloidin.

Fractionation of Cells Using Triton X-100-- After transfection, the cells (5 × 106 cells) washed with phosphate-buffered saline were incubated for 5 min at 0 °C with 0.5% Triton X-100 in 20 mM Hepes, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 2 µg/ml trypsin inhibitor and centrifuged at 12,000 × g for 3 min at 4 °C (11). The pellet was washed once with the same buffer. The supernatant and pellet were used as the Triton X-100-soluble and Triton X-100-insoluble fractions, respectively (11).

Phosphorylation of gamma 12 in Transfected Cells-- 24 h after transfection, the culture medium was replaced with DMEM for 24 h prior to the 32P incorporation experiment. The cells were washed twice with phosphate-free medium (Eagle's minimum essential medium without sodium phosphate), preincubated for 1 h at 37 °C in the same medium containing [32P]orthophosphate (0.2 mCi), and then incubated for 30 min with 10% calf serum in DMEM. After labeling, the cells were washed with the ice-cold phosphate-buffered saline and then suspended in 0.2 ml of a solution containing 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml trypsin inhibitor, 10 nM calyculin A, and 1% CHAPS. The suspension was mixed with a vortex mixer and centrifuged at 100,000 × g for 15 min at 4 °C. The supernatant was incubated at 4 °C for 2 h with 5 µg of affinity-purified antibody against gamma 7, which had been preincubated with protein A-Sepharose beads. The Sepharose beads were washed with the solution containing 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml trypsin inhibitor, 10 nM calyculin A, and 0.1% CHAPS. The beads were suspended in 30 µl of sample buffer for electrophoresis, and each resultant supernatant was subjected to Tricine/SDS-polyacrylamide gel electrophoresis (23) with subsequent autoradiography (6). For immunoblotting, cells were trypsinized 48 h after transfection, seeded into culture dishes, and incubated for 2 h at 37 °C in DMEM with 10% calf serum. Then cell lysates were subjected to Tricine/SDS-polyacrylamide gel electrophoresis for immunoblotting with the antibody against phosphorylated gamma 12 (p-gamma 12) (9).

Cell Migration Assay-- Migration through a membrane in response to serum was assayed with the use of a Chemotaxicell chamber (Kurabo) with an 8-µm polycarbonate filter (24). Transfected cells were trypsinized and counted, and 5 × 104 cells/well were loaded into the top wells in DMEM. The bottom wells were similarly filled with DMEM with 10% calf serum so that the cells were exposed to a gradient of serum factors. The chambers were incubated for 2 h at 37 °C, and the membranes were removed and stained. Under these conditions, a negligible number of cells fell off the bottom of the filter. The number of cells that had migrated through the membrane was counted for each well. To determine cell migration by chemokinesis, top and bottom wells were filled with media with 10% calf serum so that the cells were exposed to no gradient of serum factors. Cell migration in the absence of a gradient was about 40% of that in a gradient, indicating that the observed migration consisted of both chemotaxis and chemokinesis.

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Immunocytochemical double staining of normal NIH 3T3 cells with phalloidin showed the complete overlap of gamma 12 staining with staining of actin stress fibers, which were found crossing the entire cell (Fig. 1A) as previously reported for Swiss 3T3 cells (11). To investigate the role of gamma 12, we co-transfected gamma 12 or various gamma  subunits with beta 1 in NIH 3T3 cells. Most cells overexpressing gamma 12 were rounded, with disruption of F-actin architecture (Fig. 1B), whereas some cells overexpressing gamma 12 had decreased stress fibers with a flattened shape (Fig. 1C). Because the fluorescence of gamma  subunits in transfected cells was much stronger than that in normal cells, photographs with short exposure were shown in Fig. 1 (B-G). Therefore, the staining of endogenous gamma 12 in normal cells was faint or not observed in Fig. 1 (B and C). In contrast with gamma 12, overexpression of gamma 2, gamma 5, and gamma 7 did not induce such dramatic changes in cell shape, but some of cells overexpressing these gamma  subunits showed decreased stress fibers in the center and adopted a flattened, rounded shape (Fig. 1, D-F). Immunoblotting analyses showed that similar amounts of beta 1 and transfected gamma  subunits were expressed in these experiments, whereas basal levels of gamma 5 and gamma 12, major endogenous gamma  subunits in NIH 3T3 cells, were detected in all cells (Fig. 2A). To examine whether the expressed gamma  subunits associated with cytoskeletal components, transfected cells were fractionated using Triton X-100. A large portion of the expressed gamma 12 was present in the Triton X-100-insoluble fraction in transfected cells, and a similar distribution of endogenous gamma 12 was observed in control cells, whereas most gamma 2, gamma 5, and gamma 7 were present in Triton X-100-soluble fractions (Fig. 2B). These results indicate that expressed gamma 12, but not other isoforms, associates with cytoskeletal components, which is consistent with the localization of endogenous gamma  subunits in Swiss 3T3 cells (11).


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Fig. 1.   Immunocytochemistry of normal and transfected NIH 3T3 cells. A, co-localization of gamma 12 and F-actin in normal cells. NIH 3T3 cells were double stained for gamma 12 (upper panels) and F-actin (lower panels). B-F, effect of transfection of various gamma  isoforms on morphology. NIH 3T3 cells were co-transfected with beta 1 and gamma 12 (B and C), gamma 2 (D), gamma 5 (E), or gamma 7 (F) and double stained for the respective gamma  (upper panels) and F-actin (lower panels). Most cells (60-70%) overexpressing gamma 12 were rounding (B), whereas a small number of cells overexpressing gamma 12 had decreased stress fibers with a flattened shape (C). G, prevention of gamma 12-induced cell rounding by co-transfection of beta ARKct. NIH 3T3 cells were co-transfected with beta 1, gamma 12, and beta ARKct and double stained for gamma 12 (upper panel) and F-actin (lower panel). Scale bar, 50 µm.


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Fig. 2.   Expression of beta  and gamma  subunits in transfected cells and association of expressed gamma  subunits with cytoskeletal components. A, expression of beta  and gamma  subunits in transfected cells. NIH 3T3 cells were co-transfected with beta 1 and the various gamma  subunits as indicated. Equal amounts of cell lysates were subjected to Tricine/SDS-polyacrylamide gel electrophoresis for gamma  subunits or SDS-polyacrylamide gel electrophoresis for beta  subunit and immunoblotted with antibodies against beta  subunit and various gamma  subunits. The standards (Std, from top to bottom) were purified bovine beta gamma 2 (10 ng), beta gamma 5 (5 ng), beta gamma 7 (5 ng), beta gamma 12 (5 ng), and beta gamma 2 (5 ng). Because the antibody used to detect gamma 7 cross-reacted with gamma 2, gamma 3 and gamma 12 (22), staining for gamma 7 artifactually stained gamma 2 and gamma 12 as well as gamma 7. The other antibodies used to stain the gamma  subunits were specific for their respective isoforms (6, 22). B, association of transfected gamma  subunits with cytoskeletal components. Transfected cells were fractionated into Triton X-100-soluble (Sol) and -insoluble (Insol) fractions. Fractions were then subjected to immunoblotting with respective antibodies.

In contrast with the cells co-transfected with beta 1 and gamma 12, the cells transfected with gamma 12 alone were unchanged, suggesting that beta 1 and gamma 12 were co-expressed in cells and the beta 1gamma 12 complex induced cell rounding (data not shown). To test further whether the beta 1gamma 12 complex was indeed involved in induction of cell rounding, we expressed beta ARKct (19) and Gi2alpha , both of which are expected to bind and sequester free beta gamma . Co-expression of beta ARKct (Fig. 1G) or Gi2alpha (data not shown) prevented beta 1gamma 12-induced cell rounding, supporting the involvement of the beta gamma complex in these changes.

The round shape of the cells suggested a decrease of cell adhesion, which might influence cell migration (24-26). To examine this possibility, Boyden chamber cell migration assays were performed for cells transfected with various gamma  subunits. Cell migration markedly increased in cells transfected with gamma 12 but did not significantly change in cells transfected with gamma 2, gamma 5, or gamma 7 (Fig. 3). Co-transfection of beta ARKct again suppressed the increase of cell motility induced by gamma 12. These results suggested that gamma 12, but not other gamma  subunits, was involved in enhancement of cell motility. Expression of beta ARKct alone in normal cells decreased cell motility, suggesting that beta gamma 12-induced cell migration occurred in normal cells (data not shown).


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Fig. 3.   Effect of transfection of various gamma  subunits on cell migration in response to calf serum. NIH 3T3 cells were co-transfected with beta 1, various gamma  subunits, and beta ARKct, and cell migration was examined. Data represent the means ± S.E. of nine experiments.

Comparison of amino acid sequences of various isoforms of gamma  revealed diverged residues concentrated at the N-terminal region (6). To test whether the ability of gamma 12 to associate with F-actin is determined by its N-terminal sequence, the N-terminal truncated gamma 12 (gamma 12Delta N5, Fig. 4A) was transfected into NIH 3T3 cells. The deletion of the N terminus decreased the ability of gamma 12 to associate with cytoskeletal fractions (Fig. 4B), indicating that the N-terminal region of gamma 12 is important for the association with F-actin. The weak association of gamma 12Delta N5 with cytoskeletal fractions was not due to an inability to form the beta gamma complex, because gamma 12Delta N5 was coimmunoprecipitated with an antibody against Gi2alpha when co-transfected with beta  and Gi2alpha but not when co-transfected with only Gi2alpha (data not shown). The gamma 12Delta N5 mutant neither caused cell rounding nor enhanced cell migration (Fig. 4, C and F), suggesting that the association of gamma 12 with F-actin is important to induce these changes.


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Fig. 4.   Involvement of phosphorylation of gamma 12 in induction of cell rounding and increase of migration. A, the N-terminal amino acid sequences of gamma 12, gamma 2, and their mutants. B, association of gamma 12 and gamma 2 mutants with cytoskeletal components. NIH 3T3 cells were co-transfected with beta 1 and various mutants of gamma  subunits, fractionated into Triton X-100-soluble (S) and -insoluble (In) fractions, and then subjected to immunoblotting with the antibody against gamma 7, which was generated with the peptide corresponding to the C-terminal amino acid sequence and cross-reacted with various gamma  subunits, including gamma 2 and gamma 12. The fractions from cells transfected with gamma 12Delta N5 shows two bands corresponding to gamma 12Delta N5 (lower band) and endogenous gamma 12 (upper band). The intensity of each band was quantified by densitometry using the NIH Image program, and the ratios of the gamma  subunit associating with cytoskeletal fraction (In/S + In) were obtained as follows (means of two experiments): Mock, 0.32; gamma 12, 0.44; gamma 12Delta N5, 0.18; gamma 12S2A, 0.41; gamma 12S2E, 0.39; gamma 2SSK, 0.13. C, immunocytochemisty of transfected cells stained with the antibody against gamma 7. Scale bar, 50 µm. D, phosphorylation of gamma  subunits in transfected cells determined by immunoblotting with the antibody against p-gamma 12. E, phosphorylation of gamma  subunits determined by 32P incorporation. F, effect of transfection of gamma 12 and gamma 2 mutants on cell migration. Data represent the means ± S.E. of nine experiments.

On the other hand, the N-terminal region contains a site phosphorylated by PKC (6, 9), and therefore it is possible that phosphorylation of gamma 12 is involved in induction of these changes. When phosphorylation of gamma 12 was analyzed by immunoblotting with the antibody against p-gamma 12, the expressed gamma 12 was strongly phosphorylated, whereas weak phosphorylation of endogenous gamma 12 was observed (Fig. 4D). The phosphorylation of the expressed gamma 12 determined with 32P incorporation was also greater than that of the endogenous gamma 12 observed in control cells, but the increase appeared to be smaller than that observed in immunoblots (Fig. 4E). The conditions of these experiments were not identical, but the main reason for the small increase of phosphorylation was that some gamma 12 subunits in transfected cells had already been phosphorylated before the 32P incorporation experiment (data not shown). We then transfected NIH 3T3 cells with the mutant gamma 12S2A, in which Ser2, a phosphorylation site,2 is replaced with alanine (Fig. 4A). The gamma 12S2A, which was not phosphorylated, could associate with the cytoskeleton but had no effect on morphology and motility of cells (Fig. 4). Because glutamic acid can substitute for phosphoserine in some proteins activated by phosphorylation, we next transfected cells with mutant gamma 12S2E, in which Ser2 is replaced with glutamate (Fig. 4A). This mutant caused similar effects in transfected cells to wild-type gamma 12, except of the increase of phosphorylation (Fig. 4). These results supported the idea that phosphorylation of gamma 12 induced cell rounding and enhanced cell motility. In Fig. 4 (D and E), smaller increases of phosphorylation were observed in the cells transfected with gamma 12Delta N5, gamma 12S2A, and gamma 12S2E, in comparison with control cells. The reason for these increases will be discussed later (see "Discussion").

To further evaluate the effect of phosphorylation on the activity of gamma  subunits, we mutated the N-terminal region of a different gamma , gamma 2, to introduce SSK, the phosphorylation motif for PKC (gamma 2SSK, Fig. 4A) (9). The gamma 2SSK was phosphorylated as well as gamma 12 in cells when judged by 32P incorporation, whereas the antibody against p-gamma 12 weakly recognized phosphorylated gamma 2SSK, probably due to a low reactivity with the mutant. However, the gamma 2SSK, which hardly associated with cytoskeletal fractions, neither induced cell rounding nor increased cell migration (Fig. 4), suggesting that phosphorylation is not sufficient for the changes induced by phosphorylated gamma 12. The weak association of this mutant with cytoskeletal fractions (Fig. 4B) suggested that the association with the cytoskeleton might be essential for enhancing cell migration.

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The present study showed that transfection of beta gamma 12 markedly induced cell rounding and increased cell migration, but transfection of beta gamma 2, beta gamma 5, and beta gamma 7 induced only minor changes, clearly indicating a functional difference among gamma  subunits. This specific function of gamma 12 is derived from its unique property of selective phosphorylation by PKC. However, the phosphorylation is not sufficient to account for the activity of gamma 12, because the gamma 2SSK mutant, which could be phosphorylated, did not induce these changes in cells. This is in contrast to the result that the activity of the beta gamma complex containing phosphorylated gamma 10G4K (gamma 10SSK) mutant was similar to that of the phosphorylated beta gamma 12 in stimulating type II adenylyl cyclase (10). Some part(s) of the structure of gamma 12 other than the N terminus might also be important for enhancing cell migration or/and some other characteristic of gamma 12 important for associating with F-actin might be essential for inducing these changes. The present results demonstrated that gamma  subunits unable to associate with cytoskeletal factions did not increase cell migration, but further experiments are necessary to show whether association with F-actin is essential.

Although the effects of gamma 2, gamma 5, and gamma 7 were not very remarkable, decreases of stress fibers (Fig. 1, E-G) and small increases of cell motility (Fig. 3) were observed in cells transfected with these gamma  subunits. One previous report showed that transfection of beta 1gamma 2 did not induce morphological changes in Swiss 3T3 cells (27), but another report indicated that microinjection of beta 1gamma 2 reduced stress fibers in CV-1 cells (28), which is basically consistent with our present observations. These isoforms may have weak activities, but it is also possible that the apparent effects of gamma 2, gamma 5, and gamma 7 are due to beta gamma 12 released from endogenous G protein due to displacement by the overexpressed beta gamma . This speculation might be supported by the evidence that phosphorylation of gamma 12 slightly increased in the cells transfected with gamma  subunits other than wild-type gamma 12 in comparison with control cells (Fig. 4, D and E). Because similar amounts of endogenous gamma 12 exist in all these cells, the increase of phosphorylated gamma 12 suggests the replacement of the beta gamma 12 in endogenous G protein and an increase of the free beta gamma 12.

We have shown that phosphorylation of gamma 12 enhances fibroblast migration in response to serum. A variety of growth factors, including platelet-derived growth factor (26, 29), basic fibroblast growth factor (30), lysophosphatidic acid (26), and serum (24), were shown to stimulate migration of fibroblasts. Down-regulation or inhibition of PKC abolished the effect of platelet-derived growth factor and basic fibroblast growth factor on cell migration, suggesting the role of PKC in these processes (29, 30). Our previous observation that platelet-derived growth factor, basic fibroblast growth factor, lysophosphatidic acid, and serum stimulated phosphorylation of gamma 12 in Swiss 3T3 cells (9) strongly suggests the involvement of phosphorylation of gamma 12 in cell migration stimulated by these growth factors. For phosphorylation of gamma 12, activation of G proteins is important as well as PKC activation, because free beta gamma 12 was a better substrate for PKC than the trimer form (6). Lysophosphatidic acid could stimulate Gi and Gq, so that both G protein and PKC could be activated by this agonist. By contrast, receptor tyrosine kinases are well known to stimulate PKC via activation of phospholipase Cgamma , and the evidence that basic fibroblast growth factor-stimulated migration of endothelial cells was reduced by pertussis toxin suggests the involvement of G protein activation in this process (31). Neptune and Bourne (32) and Arai et al. (33) reported that expressed Gi-coupled receptors, such as D2 dopamine and opioid receptors, induced cell migration, which was prevented by beta ARKct and Gtalpha , suggesting involvement of beta gamma subunits. However, they also suggest that Gi activation is necessary but probably not sufficient for chemotaxis. Activation of PKC to phosphorylate gamma 12 might be necessary for maximal stimulation of chemotaxis.

Cell migration requires dynamic and coordinated disassembly and reassembly of stress fibers and focal adhesions, but the precise mechanisms regulating these processes are not clear (25). The present observations that overexpression of gamma 12 decreases stress fibers and induces cell rounding suggest that gamma 12 may be involved in disassembly of stress fibers. Because PKC and phosphoinositide 3-kinase, of which activation increases cell migration, have also been found to associate with the cytoskeleton (16, 17), the gamma 12-associated cytoskeleton may be easily accessed by these enzymes, facilitating their interaction. Recent studies showed the role of Rho family GTPases such as Rho, Rac, and Cdc42 in regulation of assembly and organization of the actin cytoskeleton (34). In the budding yeast Saccharomyces cerevisiae, beta gamma complex has been shown to associate with Cdc24, a guanine nucleotide exchange factor for Cdc42, suggesting a cascade from beta gamma to actin organization via Cdc42 (35). At present, this cascade has not been shown in mammalian cells, but future experiments will elucidate downstream signaling molecules linking beta gamma 12 to cell migration, possibly including this cascade.

    ACKNOWLEDGEMENTS

We thank Drs. M. I. Simon and T. Nukada for supplying the plasmids.

    FOOTNOTES

* This work was partly supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan.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. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Inst. for Developmental Research, Aichi Human Service Center, Kamiya-cho, Kasugai, Aichi 480-0392, Japan. Fax: 81-568-88-0829; E-mail: toasano{at}inst-hsc.pref.aichi.jp.

2 A phosphorylation site of gamma 12 for PKC is the first serine residue from the N terminus of gamma 12, which was previously designated Ser1 from the sequence of a mature gamma 12 protein (6, 9, 10) but is designated Ser2 in this paper.

    ABBREVIATIONS

The abbreviations used are: G protein, guanine nucleotide-binding protein; PKC, protein kinase C; DMEM, Dulbecco's modified essential medium; beta ARKct, C-terminal fragment of the beta -adrenergic receptor kinase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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