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Endocrinology, doi:10.1210/en.2007-1572
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Endocrinology Vol. 149, No. 4 1718-1727
Copyright © 2008 by The Endocrine Society

Insulin Increases Nuclear Protein Kinase C{delta} in L6 Skeletal Muscle Cells

Miriam Horovitz-Fried1, Tamar Brutman-Barazani1, Dov Kesten and Sanford R. Sampson

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel 52900

Address all correspondence and requests for reprints to: S. R. Sampson, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. E-mail: sampsos{at}mail.biu.ac.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protein kinase C (PKC) isoforms are involved in the transduction of a number of signals important for the regulation of cell growth, differentiation, apoptosis, and other cellular functions. PKC proteins reside in the cytoplasm in an inactive state translocate to various membranes to become fully activated in the presence of specific cofactors. Recent evidence indicates that PKC isoforms have an important role in the nucleus. We recently showed that insulin rapidly increases PKC{delta} RNA and protein. In this study we initially found that insulin induces an increase in PKC{delta} protein in the nuclear fraction. We therefore attempted to elucidate the mechanism of the insulin-induced increase in nuclear PKC{delta}. Studies were performed on L6 skeletal myoblasts and myotubes. The increase in nuclear PKC{delta} appeared to be unique to insulin because it was not induced by other growth factors or rosiglitazone. Inhibition of transcription or translation blocked the insulin-induced increase in nuclear PKC{delta}, whereas inhibition of protein import did not. Inhibition of protein export from the nucleus reduced the insulin-induced increase in PKC{delta} in the cytoplasm and increased it in the nucleus. The increase in nuclear PKC{delta} induced by insulin was reduced but not abrogated by treatment of isolated nuclei by trypsin digestion. Finally, we showed that insulin induced incorporation of 35S-methionine into nuclear PKC{delta} protein; this effect was not blocked by inhibition of nuclear import. Thus, these results suggest that insulin may induce nuclear-associated, or possibly nuclear, translation of PKC{delta} protein.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROTEIN KINASE C (PKC) isozymes are a family of serine/threonine kinases involved in the transduction of number signals important for the regulation of cell growth, differentiation, apoptosis, and many other functions (1, 2, 3, 4). At present, 11 isoforms have been cloned and identified. They have been classified into three main groups that share a common requirement for phospholipids for their activity but differ in structure and their dependence on other activators. Conventional PKCs ({alpha}, βI, βII,{gamma}) require phosphatidylserine (PS), Ca2+, and diacylglycerol or phorbol esters for activation. Novel PKCs ({delta}, {epsilon}, {nu}, {theta}) require diacylglycerol and PS for activation. Atypical PKCs ({zeta}, {iota}, {lambda}) require only PS for activation (5, 6, 7, 8). The biological functions of PKC have mostly been linked with events occurring at the plasma membrane or other membrane components in the cytoplasm. This is because PKCs proteins are believed to remain in an inactive, cytoskeletal-associated state in the cytoplasm; after phosphorylation, they translocate to the plasma membrane or membranes of cytoplasmic organelles to become fully activated in the presence of specific cofactors (1, 9, 10). Recent studies, however, indicate that PKC proteins may also have a role in the nucleus. Thus, it has been suggested that nuclear PKCs may participate in cascades that communicate signals that are generated at the plasma membrane and are transmitted to the nucleus (11, 12, 13). Studies have identified several proteins that can act as PKC substrates (11, 14, 15) and other nuclear PKC-binding proteins (16, 17, 18, 19). Nuclear PKCs are involved in the regulation of several important biological process such as cell proliferation [PKCβII (20, 21)], cell differentiations [PKC{alpha} (22)], neoplastic transformation [PKCβ (23, 24)], and apoptosis [PKC{alpha} and PKC{delta} (25, 26)].

We recently reported that insulin stimulation of skeletal muscle rapidly increases total PKC{delta} RNA and protein levels on a time scale of within 5 min (27). These effects are accompanied by rapid activation of the PKC{delta} promoter and are abrogated by pretreatment with inhibitors of either translation [cycloheximide (CH)] or transcription [actinomycin D (AD) and 5,6-dichlorobenzimidazole riboside (DRB)]. Thus, insulin induces de novo PKC{delta} protein synthesis as well as transcription of new PKC{delta} RNA. During the course of these studies, we examined the possibility that elevations in total cytosolic PKC{delta} might result from translocation from other subcellular fractions. To our surprise, our results indicated that the ability of insulin to induce an increase in PKC{delta} protein occurred with the same rapid time course in the nucleus as it does in the cytoplasmic and cytoskeletal fractions, i.e. within 5 min of insulin stimulation. These observations suggested that insulin may induce rapid translocation of nascent PKC{delta} into the nucleus or de novo synthesis of the PKC{delta} protein itself within or associated with the nucleus.

According to accepted paradigm, polymerase II transcribes DNA to mRNA; the RNA then translocates to the cytoplasm in which it is translated to protein by the ribosomes. Synthesis of protein in the nucleus is considered unlikely, despite occasional reports that suggest the existence of this phenomenon (28, 29, 30, 31). These studies performed on HeLa cells showed that whereas most of protein translation occurs in the cytoplasm, some (10–15%) protein translation may occur in the nucleus. Nuclear translation has been refuted by other studies (32, 33, 34), in which it was concluded that if nuclear translation occurs, it would be limited to less than 1% of the total protein.

In the current study, we examined the mechanisms underlying the rapid increase in nuclear PKC{delta} in response to insulin of skeletal muscle. We found that the effect appeared to be limited to insulin and was not blocked by inhibitors of nuclear import or export. Our data suggest that after insulin stimulation, PKC{delta} may be both transcribed and translated within the cell nucleus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue culture media and serum were purchased from Biological Industries (Beit HaEmek, Israel). Enhanced chemical luminescence was performed using antibodies purchased from Bio-Rad (Hercules, CA) and other reagents from Sigma (St. Louis, MO). The following antibodies were used: anti-PKC{delta} and anti-PKC{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA); antiskeletal muscle β-actin (Sigma); horseradish peroxidase-conjugated antirabbit and antimouse IgG (Bio-Rad); Alexa fluor488 goat antirabbit IgG was purchased from Invitrogen (Eugene, OR).

Leupeptin, aprotinin, phenylmethylsulfonyl fluoride, dithiothreitol, orthovanadate, pepstatin, cyclohexamide, DRB, AD, and wheat germ agglutinin (WGA) were purchased from Sigma. Insulin (HumulinR, recombinant human insulin) was purchased from Lilly France SA (Fergersheim, France). Leptomycin B (LMB) was purchased from Calbiochem (La Jolla, CA). 35S-methionine was purchased from PerkinElmer (Boston, MA).

Cell culture
L6 cells were grown in {alpha}MEM supplemented with 10% fetal calf serum for 4 d after confluence, with media changed daily; cells were allowed to differentiate spontaneously or induced by changing the media to one supplemented with 2% fetal calf serum. In some experiments (immunostaining), studies were done on L6 myoblasts, which are also used as a model system for insulin signaling (35, 36)

Cytoplasmic extract
Dishes were washed with Ca2+/Mg2+-free PBS and then mechanically detached with cell scraper in radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.4); 150 mM NaCl; 1 nM EDTA; 10 mM NaF; 1% Triton X-100; 0.1% sodium dodecyl sulfate; 1% Na deoxycholate] containing a cocktail of antiproteases and antiphosphatases (Sigma). After scraping, the preparation was centrifuged at 20,000 x g for 20 min at 4 C. The supernatant from this step was designated the cytoplasmic fraction.

Nuclear extracts
Nuclear extracts were prepared as described (35). Dishes were washed with Ca2+/Mg2+-free PBS and then mechanically detached with a cell scraper in PBS. Cells were transferred to Eppendorf tubes and centrifuged at 200 x g (1500 rpm) for 15 min at 4 C. The pellet was resuspended in buffer [1 M Tris (pH 7.5), 2.5 M NaCl, 2 mM EDTA, 1 mM EGTA] containing a cocktail of protease and phosphatase inhibitors (Sigma). The suspension was then homogenized in a Dounce glass homogenizer (30 strokes) and centrifuged at 400 x g (2000 rpm) for 15 min at 4 C. The pellet was resuspended again in buffer containing cocktails of protease and phosphatase inhibitors and centrifuged at 400 x g for 15 min at 4 C. The pellet from this centrifugation was suspended in Buffer 1 (0.5 M sucrose; 5 mM MgCl2; 0.1 mM EDTA; 10 mM Tris, pH 8; 1 mM dithiothreitol) and centrifuged at 23,100 x g for 45 min at 4 C. Purification of the nuclear preparation (Fig. 1Go) was verified by scanning electron microscopy (SEM) at the step prior to disruption of the nuclei and by Western blotting for tubulin (a cytoplasmic marker) and as well as for actin (to control for equal loading of protein). The pellet from this step was resuspended in buffer 2 (buffer 1 without EGTA, with 0.6 M KCl) and left in ice for 30 min. The suspension was then centrifuged at 75,500 x g for 60 min at 4 C. The supernatant from this step was designated the nuclear fraction.


Figure 1
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  		FIG. 1. Western blot and photomicrograph showing purity of nuclear preparations. Differentiated myotubes were subjected to cell fractionation, SDS-PAGE, and transfer, as described in Materials and Methods. A, Transferred proteins were immunoblotted (IB) with antibodies to tubulin [cytoplasmic marker (Cyto)] and actin (to verify equal loading). The Western blots are from two separate experiments. Tubulin is undetectable in the nuclear fraction (Nuc). B, Transferred proteins were immunoblotted with antibodies to histone H3 (nuclear marker) and actin (to verify equal loading). The Western blots are from two separate experiments. Histone is undetectable in the cytoplasmic fraction. C, Photographs (SEM) of nuclei isolated from L6 myotubes. No cellular debris is detectable.

 
Western blot analysis
Western blots were performed as described (38, 39, 40, 41). Equal protein loading of Western blots was confirmed by immunoblotting for skeletal muscle β-actin.

RT-PCR
Total RNA was obtained using RNeasy minikit (QIAGEN, Valencia, CA) from control and insulin stimulated cells. Reverse transcription was performed on 1 µg total RNA using One Tube RT-PCR system (Roche, Mannheim, Germany) and 10 µM specific primers for PKC{delta} and S12 control (rRNA, housekeeping gene). The reverse transcription reaction was amplified for 40 cycles (94 C, 1 min; 60 C, 1 min; 72 C, 1 min; 70 C, 10 min). Finally, 50% of the amplified products were resolved on a 1% agarose gel. Specific primers for PKC{delta} and S12 were designed based on reported sequences as described (27).

Cell preparation for immunofluorescence imaging
Cells were seeded on coverslips at 3 x 105 cells/well in six-well plates. After appropriate treatment, cells were fixed with 4% paraformaldehyde and dried. Coverslips were washed three times, permeabilized (isotonic PBS buffer, 10% BSA, 1% Triton X-100), and incubated with PKC{delta} primary antibody for 1 h. After further wash steps, cells were stained with a fluorescent-conjugated secondary antibody, washed, mounted, and imaged under a fluorescent microscope at x400 magnification.

35S-methionine uptake
Culture dishes (90 mm; Nunc, Glostrup, Denmark) containing the muscle cells were transferred to methionine-free medium for 3 h. The medium was then changed to medium containing 35S-methionine (40 µCi/ml) for 5–15 min after treatment with insulin for 5–15 min. Cells were washed three times with PBS, scraped, and lysates prepared for immunoprecipitation with specific antibody to PKC{delta}, as described below. The immunoprecipitates were then subjected to SDS-PAGE. The resulting gel containing immunoprecipitated PKC{delta} was exposed to x-ray film at –70 C for 2 wk.

Immunoprecipitation
Specific antibody to PKC{delta} (dilution 1:100) was added to 400 µg protein from the nuclear extract and was rotated continuously for 60 min at 4 C. After the samples were rotated for 60 min, 30 µl of A/G Sepharose was added and rotated overnight at 4 C. The samples were then centrifuged at 2000 x g for 10 min at 4 C, and the pellet was washed three times with buffer 2 by centrifugation at 2000 x g for 2 min at 4 C. The pellet was then resuspended in 25 µl of sample buffer [0.5 M Tris HCl (pH 6.8); 10% sodium dodecyl sulfate; 10% glycerol; 4% 2-β-mercaptoethanol; 0.05% bromophenol blue]. The suspension was again centrifuged at 500 x g (at 4 C for 10 min), boiled for 5 min, and then subjected to SDS-PAGE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Insulin increases PKC{delta} protein levels in the nucleus of L6 skeletal muscle cells
We recently reported that insulin induces an increase in total PKC{delta} protein levels by a process involving both transcription of RNA and translation into new protein (27). These experiments were done on whole-cell lysates of a variety of skeletal muscle cell preparations. In the current study, we initially attempted to determine whether the newly synthesized PKC{delta} might occur in, or be targeted to, a particular cell fraction. Accordingly, we performed studies in which PKC{delta} levels were examined in the membrane cytosolic and nuclear fractions in control and insulin-stimulated cells. As shown in Fig. 2AGo, insulin induced a rapid increase, within 5 min, in PKC{delta} protein in each of the fractions tested, membrane, cytosolic, and nuclear. The increase in nuclear PKC{delta} was also verified by confocal microscopy. As shown in Fig. 2BGo, PKC{delta} levels increased dramatically in both the nuclei and surrounding cytoplasm after insulin stimulation for 5 and 15 min. In contrast, insulin induced an increase in cytosolic, but a decrease in nuclear, PKC{alpha} protein levels (Fig. 2CGo).


Figure 2
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  		FIG. 2. Insulin increases PKC{delta} protein in the cytoplasmic (Cyto), membrane (Mem), and nuclear (Nuc) fractions of L6 skeletal muscle. Differentiated myotubes were treated with insulin (10–7 M) for 5 min. After cell fractionation, SDS-PAGE, and transfer, lysates were probed with specific antibodies against PKC{delta}. A, Western blots of insulin (Ins)-induced increase PKC{delta} protein in all three fractions. IB, Immunoblot. B, Photographs of immunostained L6 myoblasts showing effect of insulin on PKC{delta} protein levels in the cytoplasm and nucleus. C, Western blot showing that insulin-induced an increase in cytoplasmic PKC{alpha} and a decrease in nuclear PKC{alpha}. Differentiated myotubes were treated with insulin (10–7 M) for 5 min. After cell fractionation, SDS-PAGE, and transfer, proteins were probed with specific antibodies against PKC{alpha}.

 
The ability to increase nuclear PKC{delta} is unique to insulin
The question arises as to whether the effect to increase nuclear PKC{delta} might be induced by other growth factors and agents with insulin-like actions or whether the increase in nuclear PKC{delta} might be unique to insulin. To accomplish this, we examined effects of agents known to produce insulin-like responses or activate common enzymes in the insulin signaling pathway in skeletal muscle. We studied two categories of agents: 1) growth factors, such as IGF-I, epithelial growth factor, and nerve growth factor, all activators of receptor tyrosine kinases. Indeed, IGF-I is known to signal via shared elements in the insulin receptor (IR) signaling cascade and activate the IR β-subunit tyrosine kinase; and 2) rosiglitazone (RG), a member of the thiazolidinedione family of oral antidiabetic agents, which improves insulin sensitivity and glucose homeostasis in type 2 diabetic patients as well as various animal models of diabetes and obesity. Neither IGF-I (27) nor any of the other growth factors increased PKC{delta} protein levels, in either total cell lysates or any subcellular fractions (not shown). On the other hand, RG increased total PKC{delta} levels within 5 min, similar to the effects of insulin (Fig. 3AGo). To investigate further the mechanism by which RG increases PKC{delta} protein, we pretreated cells with inhibitors of transcription or translation. Interestingly, pretreatment of cells with CH abrogated the RG-induced increase in PKC{delta} protein, whereas the effect of RG was not altered by pretreatment with AD (Fig. 3BGo), an inhibitor of transcription. These findings indicated that RG increases translation of PKC{delta} RNA into protein but does not induce an increase in PKC{delta} RNA. Furthermore, this increase in PKC{delta} protein occurred only in the cytoplasm (Fig. 3BGo) and not in the nucleus (Fig. 3CGo). This was further confirmed in studies on PKC{delta} RNA in which we found that RG, in contrast to insulin, did not increase PKC{delta} RNA levels (Fig. 3DGo).


Figure 3
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  		FIG. 3. RG increases PKC{delta} protein in the cytoplasm but not nuclear PKC{delta} or PKC{delta} RNA in skeletal muscle. Differentiated myotubes were treated with RG (10–6 M) for the times indicated. Cell fractionation, SDS-PAGE, and transfer were performed as in Fig. 2Go. A, Western blots showing effects of insulin (Ins) and RG on cytosolic PKC{delta} protein levels. The graph to the right shows densitometry measurements of the Western blots. IB, Immunoblot. B, Western blot of cytosolic (Cyto) PKC{delta} in control (Con) and RG-stimulated cells. The blot shows that CH (1 mM, 12 h) but not AD (1 µg/ml, 3 h) abrogates RG-induced increase in cytosolic PKC{delta} protein. C, Nuclear PKC{delta} protein is not increased by RG. Nuclear fractions were prepared from control and RG-stimulated cells as described in Materials and Methods. After cell fractionation, SDS-PAGE, and transfer, nuclear proteins were probed with specific antibodies against PKC{delta}. D, PKC{delta} RNA is not increased by RG. RNA was extracted from control and RG-stimulated cells, and RT-PCR was performed as described in Materials and Methods.

 
The insulin-induced increase in nuclear PKC{delta} protein is abrogated by inhibitors of transcription and translation
Our findings indicate that the ability to induce a rapid increase in nuclear PKC{delta} protein is unique to insulin and is closely linked in time to PKC{delta} RNA transcription. One possible mechanism for the increase in nuclear PKC{delta} is its translocation to the nucleus either from an existing pool or newly synthesized PKC{delta} protein in the cytoplasm. Because we have previously shown that insulin induces rapid de novo synthesis of PKC{delta} protein (27), we first investigated the effects of inhibition of translation (CH) or transcription (AD, DRB) on the insulin-induced increase in nuclear PKC{delta} protein. Cells were treated with CH, AD, or DRB before stimulation with insulin. Nuclear fractions were then prepared and subjected to SDS-PAGE and Western blotting. In addition, cells were fixed on coverslips for fluorescence microscopy (as described in Materials and Methods). Figure 4Go shows results obtained in these experiments. Pretreatment of L6 cells with CH almost completely abrogated the insulin-induced increase in nuclear PKC{delta} protein, thus indicating that increased PKC{delta} protein in the nucleus derives from a newly synthesized pool. These results are further strengthened by studies in which cells were treated with inhibitors of transcription (AD or DRB), which also blocked the insulin-induced increase in nuclear PKC{delta} (Fig. 5Go).


Figure 4
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  		FIG. 4. Inhibition of translation blocks the insulin-induced increase in nuclear PKC{delta}. Differentiated myotubes were pretreated with CH (3 h, 1 mM) and then with insulin (Ins; 10–7 M) for 5 min. Nuclear extracts were prepared as described in Materials and Methods. After SDS-PAGE and transfer, nuclear proteins were probed with specific antibodies against PKC{delta}. A, Western blots showing the ability of CH to block insulin-induced increase in nuclear PKC{delta} protein. IB, Immunoblot. B, Graph displaying results of densitometry measurements made on Western blots; each bar represents the mean ± SE of the measurements of the ratio of PKC{delta} to actin in three separate experiments (*, P < 0.005 vs. control). C, Photographs of immunostained L6 myoblasts showing the blocking by CH of the insulin-induced increase in PKC{delta} protein levels in the nucleus.

 

Figure 5
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  		FIG. 5. Inhibition of transcription blocks insulin-induced increase in nuclear and cytoplasmic PKC{delta} protein. Differentiated myotubes were pretreated with AD (3 h, 1 µg/ml) or DRB (1 h, 50 µg/ml) and then with insulin (Ins; 10–7 M) for 5 min. Nuclear extracts were prepared as described in Materials and Methods. After SDS-PAGE and transfer, proteins were probed with specific antibodies against PKC{delta}. A, Western blots showing the ability of AD to block insulin-induced increase in nuclear PKC{delta} protein. IB, Immunoblot. B, Graph displaying results of densitometry measurements made on Western blots; each bar represents the mean ± SE of the measurements of the ratio of PKC{delta} to actin in three separate experiments (*, P < 0.005 vs. control). C, Photographs of immunostained L6 myoblasts showing effect of DRB to inhibit insulin-induced increase in PKC{delta} protein levels in the nucleus.

 
Inhibition of nuclear import does not abrogate the insulin-induced increase in PKC{delta} protein levels in the nucleus
The results so far are consistent with the possibility that the insulin-induced increase in nuclear PKC{delta} protein may result from rapid synthesis followed by translocation into the nucleus. To investigate this possibility, we treated cells with WGA (100 mg/ml, 3 h), an inhibitor of nuclear import (42, 43, 44) before insulin stimulation. Treatment with WGA caused an increase in PKC{delta} in the absence of insulin treatment, but to our surprise, inhibition of nuclear import did not have any effect on the insulin-induced increase in nuclear PKC{delta} (Fig. 6Go, A and C). In contrast, this treatment did inhibit insulin-induced translocation of specificity protein-1 from the cytoplasm to the nucleus (Fig. 6BGo; see Ref. 42). This results in a slight reduction in the insulin-induced increase in cytosolic PKC{delta} (42). The insulin-induced decrease in level of nuclear PKC{alpha} was essentially unaffected by treatment with WGA (Fig. 6AGo). It remains possible that PKC{delta} might be translated on the nuclear envelope in which it remains bound. To examine this possibility, we isolated nuclei from insulin-stimulated and unstimulated cells and treated the isolated nuclei with trypsin (1 mg/ml) to dislodge any PKC{delta} bound to the nuclear envelope. As can be seen in Fig. 6DGo, the insulin-induced increase in PKC{delta} was maintained but reduced slightly in trypsin-treated isolated nuclei.


Figure 6
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  		FIG. 6. Inhibition of protein import does not reduce and slightly increases the induction by insulin of PKC{delta} protein in the nuclear fraction. Differentiated myotubes were pretreated with import inhibitor (WGA, 1 h, 100 µg/ml) and then with insulin (Ins; 10–7 M) for 5 min. Nuclear extracts were prepared as described in Materials and Methods. After SDS-PAGE and transfer, proteins were probed with specific antibodies against PKC{delta} or PKC{alpha}. A, Western blots showing that after treatment with WGA, basal and insulin-induced increase in nuclear PKC{delta} was increased. In contrast, WGA did not alter the effect of insulin to decrease nuclear PKC{alpha} levels. The graph displays results of densitometry measurements made on Western blots; each bar represents the mean ± SE of the measurements of the ratio of PKC{delta} to actin in three separate experiments (*, P < 0.005 vs. control). IB, Immunoblot. B, Western blots showing that after treatment with WGA, the level of specificity protein-1 (SP-1) in basal and insulin-stimulated cells was reduced in the nuclear fraction. C, Photographs of immunostained L6 myoblasts showing effects of inhibition of nuclear import on insulin-induced increase in nuclear PKC{delta} protein. D, Trypsin digestion of proteins from the nuclear envelope does not abrogate the insulin-induced increase in nuclear PKC{delta} protein. Differentiated myotubes were treated with insulin (10–7 M) for 5 min. After cell fractionation nuclei were treated or not with trypsin as described in Materials and Methods. After SDS-PAGE and transfer, proteins were probed with specific antibodies against PKC{delta}.

 
Inhibition of nuclear export increases the insulin-induced increase in PKC{delta} protein levels in the nucleus and reduces the insulin-induced increase in PKC{delta} protein levels in the cytoplasm
The results so far show that the insulin-induced increase in nuclear PKC{delta} protein does not appear to be explained by translocation from the cytoplasm to the nucleus. This leads to the possibility that PKC{delta} may be translated in or associated with the nucleus in response to insulin. We attempted to further rule out the possibility of nuclear translation by inhibition of protein export. We reasoned that, after insulin induction of PKC{delta} RNA transcription, PKC{delta} RNA may migrate from the nucleus to the cytoplasm in which it is translated into PKC{delta} protein, which is then rapidly translocated back into the nucleus. Blockade of nuclear export would inhibit the export of any PKC{delta} protein (to the extent that it is synthesized or preexists in the nucleus) but allow the newly synthesized PKC{delta} RNA to migrate to the cytoplasm and undergo translation into new PKC{delta} protein. Assuming that insulin induces an increase in PKC{delta} synthesis in the cytoplasm and that the increase in nuclear PKC{delta} results from its translocation from the cytoplasm to the nucleus, inhibition of nuclear export should not affect the insulin-induced increase in cytoplasmic PKC{delta}. We studied this possibility by treating unstimulated and insulin-stimulated cells with LMB (20 ng/ml, 2 h before addition of insulin), an inhibitor of nuclear export (45, 46). The results of these studies are shown in Fig. 7Go. In fact, inhibition of nuclear export reduced the insulin-induced increase in cytoplasmic PKC{delta} protein and further increased the insulin-induced PKC{delta} protein level in the nucleus (Fig. 7Go, A, B, and D). In contrast, the insulin-induced decrease in PKC{alpha} was eliminated by treatment with LMB (Fig. 7CGo).


Figure 7
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  		FIG. 7. Inhibition of nuclear export reduces the insulin-induced increase in cytosolic PKC{delta} levels and increases the insulin-induced increase in nuclear PKC{delta} protein. Differentiated myotubes were pretreated with the nuclear export inhibitor, LMB (2 h, 20 ng/ml) and then with insulin (Ins; 10–7 M) for 5 min. Nuclear and cytosolic extracts were prepared as described in Materials and Methods. After SDS-PAGE and transfer, proteins were probed with specific antibodies against PKC{delta}. A, Blockade of nuclear export reduces insulin-induced increase in cytosolic PKC{delta}. IB, Immunoblot. B, Western blots showing that inhibition of nuclear export increases PKC{delta} protein and slightly elevates insulin-induced increase in nuclear PKC{delta} protein. The graph to the right displays results of densitometry measurements made on Western blots; each bar represents the mean ± SE of the measurements of the ratio of PKC{delta} to actin in three separate experiments (*, P < 0.005 vs. control). C, Western blots showing that inhibition of nuclear export blocks insulin-induced decrease in PKC{alpha} protein. D, Photographs of immunostained L6 myoblasts showing effect of nuclear export inhibition on insulin-induced increase in nuclear PKC{delta} protein levels.

 
Insulin increases 35S-methionine incorporation into nuclear PKC{delta} protein
The results so far appear to indicate that PKC{delta} protein, once rapidly elevated in the nucleus, is closely time linked to translocation from the nucleus to the cytoplasm. As we have just shown, inhibition of nuclear export reduced insulin-induced increase in cytosolic PKC{delta} and increased insulin-induced nuclear PKC{delta}. These results indicate the possibility of nuclear-associated or nuclear translation of PKC{delta} protein. To further investigate this last hypothesis, we labeled the cells with radioactive 35S-methionine (40 mCi/ml) and examined the uptake of 35S-methionine into nuclear PKC{delta} of control and insulin-stimulated cells. As can be seen in Fig. 8AGo, insulin induced an increase in 35S-methionine incorporation into PKC{delta} protein, which rapidly increases in the nucleus. These data appear to suggest the possibility that PKC{delta} protein may indeed be translated in or associated with the nucleus. Treatment with WGA (nuclear import inhibitor) did not prevent the effect of insulin on35S-methionine incorporation into nuclear PKC{delta} protein (Fig. 8BGo).


Figure 8
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  		FIG. 8. Insulin increases 35S-methionine incorporation into nuclear PKC{delta} protein. Differentiated myotubes were labeled with 35S-methionine (40 µCi/ml) as described in Materials and Methods. The cells were treated with insulin (Ins; 10–7 M) for 5 min, after which a nuclear extract was prepared. PKC{delta} was immunoprecipitated (IP) from nuclear extracts with anti-PKC{delta} antibodies as described in Materials and Methods and then subjected to SDS-PAGE and autoradiography. B, Insulin-induced increase 35S-methionine incorporation into nuclear PKC{delta} is not blocked by inhibition of nuclear import. Differentiated myotubes were pretreated with import inhibitor (WGA, 1 h, 100 µg/ml) and then with insulin (10–7 M) for 5 min. Nuclear extracts were prepared as described in Materials and Methods. PKC{delta} was immunoprecipitated from nuclear extracts with anti-PKC{delta} antibodies as described in Materials and Methods and then subjected to SDS-PAGE and autoradiography.

 
Insulin increases PK{delta} protein in isolated, live nuclei in vitro
The results indicate that insulin increases nuclear PKC{delta} protein via a mechanism independent of nuclear import or export. This suggests that the effect may occur directly in the nucleus or on nuclear-associated elements (e.g. nuclear envelope associated proteins). To examine this further, we performed studies on isolated nuclei stimulated in vitro. Nuclei were prepared as described in Materials and Methods. Results are shown in Fig. 9Go, A and B. Figure 9AGo is a Western blot showing that insulin administered directly to the nuclei caused an increase in nuclear PKC{delta}. These results were confirmed by confocal microscopy, as shown in Fig. 9BGo.


Figure 9
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  		FIG. 9. Insulin increases PKC{delta} protein levels in isolated, live nuclei in vitro. Nuclear fraction and fraction of live nuclei were prepared as described in Materials and Methods. A, Western blots showing that insulin (Ins) induces an increase in PKC{delta} protein levels from isolated live nuclei in vitro as well as in nuclear fraction. IB, Immunoblot. B, Photographs of immunostained isolated nuclei showing the effect of insulin on PKC{delta} protein levels. The green (left lane) represents the levels of PKC{delta} and the red (middle lane) is immunostaining of the nuclei with propidium iodide. In the right lane are shown merged photographs demonstrating PKC{delta} within the nucleus (orange).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we report that insulin stimulation of skeletal muscle induces a rapid increase in PKC{delta} protein in the nuclear fraction of cell lysates. In contrast, insulin decreases PKC{alpha} protein levels in the nucleus. RG, which does increase total PKC{delta}, does not increase PKC{delta} RNA or nuclear PKC{delta} protein. These results are consistent with our recent report that showed that insulin induces a rapid increase in total PKC{delta} protein and RNA (27). Recently evidence has accumulated to show that some PKC isoforms are translocated to the nucleus in response to certain stimuli (11, 13). Several reports suggest that nuclear PKCs have a role in cascades that communicate signals generated at the plasma membrane and transmitted to the nucleus (47). Most PKC isoforms have been found to contain a nuclear localization sequence, and studies have shown that the PKC{delta} nuclear localization sequence is highly conserved in this kinase family (12). We showed previously that inhibition of translation and transcription of the cells blocked the insulin-induced increase in PKC{delta} RNA and protein (27).

We attempted to elucidate the mechanism by which insulin induces a rapid increase in nuclear PKC{delta} protein. We first examined the effect of translation inhibition on the insulin-induced increase in nuclear PKC{delta} protein levels. An inhibitor of translation (CH) blocked the insulin-induced increase in nuclear PKC{delta} protein levels. This blockade might occur as a result of inhibition of PKC{delta} translation in the cytoplasm. After treatment with cycloheximide, less PKC{delta} is found in the cytoplasmic fraction, and therefore, less protein would be translocated to the nucleus. As expected, inhibition of transcription (AD) also blocked the insulin-induced increase in nuclear PKC{delta} protein levels. Because of the inhibition of transcription, less PKC{delta} is transcribed; less mRNA of PKC{delta} is translated into protein; and, therefore, less PKC{delta} would be translocated to the nucleus.

To demonstrate that PKC{delta} protein is indeed translocated from the cytoplasm fraction to the nuclear fraction, we performed studies in which protein import into the nucleus was inhibited by treatment with a protein import inhibitor. We found that inhibition of protein import into the nucleus did not block or even reduce the insulin-induced increase in nuclear PKC{delta} protein. Interestingly, blockade of nuclear import induced an increase in nuclear PKC{delta}, even in the absence of insulin stimulation. We can only speculate as to the mechanism of this effect. WGA would be expected to inhibit import of many proteins into the nucleus. This could include factors that may down-regulate PKC{delta} (as well as other proteins) synthesis or degradation. In any case, our data are strongly suggestive that insulin induces PKC{delta} synthesis associated with (or within) the nucleus. Alternatively, the effect might be related to the ability of WGA to mimic certain effects of insulin stimulation (48). Clearly, additional studies are necessary to clarify this effect further.

In current studies (Attali, V., M. Horovitz-Fried, T. Brutman-Barazani, and S. Sampson, studies in progress) in which we are investigating effects of thiazolidinediones (rosiglitazone and troglitazone) on PKC{delta} expression in skeletal muscle, we have observed that these compounds caused an increase in protein levels of PKC{delta} but not other PKC isoforms. Several relatively recent studies have shown that these compounds have effects that, whereas in a sense positively up-regulate IR signaling, may appear to be independent of peroxisomal proliferator-activated receptor-{gamma}-related actions (36, 49, 50, 51). In this study, we also examined the possibility that RG might also increase nuclear PKC{delta} protein. We found, however, that the effect of RG appeared to occur only in the cytoplasm. Thus, the effect of insulin to increase nuclear PKC{delta} would appear to be selective for this hormone. It is also important to emphasize that the effect of insulin also appears to be selective for PKC{delta} because levels of PKC{alpha} were decreased by insulin; we have not observed effects of insulin on any other PKC isoforms.

The traditional view of transcription/translation in eukaryotes is that polymerase II transcribes the DNA to mRNA in the nucleus; the mRNA is translocated to the cytoplasm, in which it is translated to protein by the ribosomes. Synthesis of protein in the nucleus has been considered unlikely, despite occasional reports that appear to demonstrate the existence of this phenomenon (28, 29, 30, 31). Most reports agree that, whereas most of the cellular protein is translated in the cytoplasm, it has been estimated that at most as much as 1% of the proteins in the cell may be translated in the nucleus (32, 33, 34). There is both direct and indirect evidence for translation of proteins in the nucleus occurring immediately after transcription, such as occurs in bacteria. Studies with labeled amino acids have shown that, indeed, most of the labeled residues are incorporated into cytoplasmic proteins, but a small fraction of the labeled amino acids may be incorporated into polypeptides in the nucleus (30). Labeling the cells with florescent lysine showed that the accumulation of the florescence in the nucleus was time dependent and sensitive to eukaryotic but not to bacterial translation inhibitors. The translation sites that were labeled by the lysine were not scattered in the nucleus but overlapped with the transcription sites. All the factors necessary for the translation process have been found in the nucleus. These findings provide indirect evidence for the possibility of nuclear translation. On the other hand, there are several studies that refute this possibility based mainly on the objection that the nuclear preparations could have been contaminated with cytoplasmic components (32, 33, 34). It should be pointed out, however, that even these studies left open the possibility that some cellular protein translation might occur in the nucleus. We did not find evidence for contamination of the nuclear preparations in this study with cytoplasmic components. SEM examination indicated that the fraction was comprised only of nuclei; no other cellular debris was detected, and Western blotting studies showed that tubulin, a cytoplasmic protein, was undetectable in the nuclear fraction.

We attempted to further rule out the possibility of nuclear translation by inhibition of protein export. We reasoned that after insulin induction of PKC{delta} transcription, PKC{delta} RNA would migrate from the nucleus to the cytoplasm in which it is translated into PKC{delta} protein, which is then translocated into the nucleus. Blockade of nuclear protein export would inhibit the export of any PKC{delta} protein (to the extent that it is synthesized or preexists in the nucleus) but allow the newly synthesized PKC{delta} RNA to migrate to the cytoplasm and undergo translation into new PKC{delta} protein. Assuming that insulin induces an increase in PKC{delta} synthesis in the cytoplasm and that the increase in nuclear PKC{delta} results from its translocation from the cytoplasm to the nucleus, inhibition of nuclear export was not expected to affect the insulin-induced increase in cytoplasmic PKC{delta}. In fact, inhibition of nuclear export reduced the insulin-induced increase in cytoplasmic PKC{delta} protein and increased even more the insulin-induced PKC{delta} protein level in the nucleus. Similarly, inhibition of nuclear import did not reduce the ability of insulin to increase nuclear PKC{delta} protein. These findings would appear to be consistent with a nucleus-associated origin of the newly translated protein.

These results indicate that it is possible that PKC{delta} protein may belong to a group of proteins that represent a very small percentage of total cell proteins whose translation takes place in close proximity to, or possibly within, the nucleus immediately after transcription. To directly examine this possibility, we labeled cells with 35S-methionine and showed that insulin induced a rapid (within 5 min) increase in 35S-methionine incorporation into nuclear PKC{delta} protein. This effect persisted despite pretreatment of the cells with WGA, which inhibits nuclear import. Thus, all these results indicate that insulin induces nuclear-associated translation of PKC{delta} protein.

In conclusion, this study suggests the possibility that PKC{delta} protein may be translated in skeletal muscle cells in response to insulin, closely time linked to its transcription in the nucleus. This possible mechanism might explain the rapid increase in PKC{delta} protein in the nuclear fraction. The idea of rapid transcription and translation of proteins is highly unconventional and runs counter to current concepts of protein synthesis, as has been discussed (32). However, this mechanism may be unique for insulin and PKC as the data in our study appear to indicate. Insulin is released from pancreatic β-cells in a pulsatile, periodic manner, sometimes as frequently as every 20–30 min, depending on fluctuations in blood sugar levels. Studies have shown that certain PKC isoforms, PKC{delta} in particular, play a key role in the initial steps of IR signaling and may participate in IR internalization and tyrosine phosphorylation (39). Thus, it should not be surprising that expression of PKC isoforms, such as PKC{delta}, may be regulated in a rapid manner at multiple levels to sustain these kinases at effective concentrations during the course of insulin action in insulin target tissues. One of these mechanisms might include rapid and closely linked nuclear transcription and translation.


   Footnotes
 
This work was supported in part by the Russell Berrie Foundation and D-Cure, Diabetes Care in Israel, grants from the Chief Scientist’s Office of the Israel Ministry of Health, and the Sorrell Foundation.

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 27, 2007

1 M.H.-F. and T.B.-B. contributed equally to this study. Back

Abbreviations: AD, Actinomycin D; CH, cycloheximide; DRB, 5,6-dichlorobenzimidazole riboside; IR, insulin receptor; LMB, leptomycin B; PKC, protein kinase C; PS, phosphatidylserine; WGA, wheat germ agglutinin.

Received November 15, 2007.

Accepted for publication December 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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