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Molecular Endocrinology, doi:10.1210/me.2006-0193
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Molecular Endocrinology 20 (11): 2890-2897
Copyright © 2006 by The Endocrine Society

Phosphatidylinositol 4-Kinase Type II{alpha} Is Targeted Specifically to Cellugyrin-Positive Glucose Transporter 4 Vesicles

Zhao Xu, Guanrong Huang and Konstantin V. Kandror

Boston University School of Medicine, Boston, Massachusetts 02118

Address all correspondence and requests for reprints to: K. V. Kandror, Boston University School of Medicine, Department of Biochemistry, K124D, 715 Albany Street, Boston, Massachusetts 02118. E-mail: kandror{at}biochem.bumc.bu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Phosphoinositides now emerge as important regulators of membrane traffic. In particular, phosphatidylinositol 4-phosphate may serve as a precursor for polyphosphorylated derivatives of phosphatidylinositol and, also, may regulate vesicular traffic by recruiting specific proteins to the membrane. Early results have demonstrated the presence of phosphatidylinositol 4-kinase (PI4K) activity in glucose transporter 4 (Glut4) vesicles from fat and skeletal muscle cells. However, the molecular identity of phosphatidylinositol 4-kinase(s) associated with Glut4 vesicles has not been characterized. It has also been determined that Glut4 vesicles are not homogeneous and represent a mixture of at least two vesicular populations: ubiquitous cellugyrin-positive transport vesicles and specialized cellugyrin-negative insulin-responsive Glut4 storage vesicles, which are different in size, protein composition, and functional properties. Using sequential immunoadsorption, subcellular fractionation, and immunofluorescence staining, we show that virtually all PI4K activity in Glut4 vesicles is represented by PI4K type II{alpha}, which is associated with cellugyrin-positive vesicles and is not detectable in the Glut4 storage vesicles. The unique N terminus of PI4K type II{alpha} is required for the targeting of the enzyme to cellugyrin-positive vesicles. Knockdown of PI4K type II{alpha} with the help of short hairpin RNA does not decrease the amount of cellugyrin-positive vesicles in human embryonic kidney 293 cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INSULIN STIMULATES GLUCOSE uptake in fat and skeletal muscle cells by promoting fusion of intracellular glucose transporter 4 (Glut4)-storage vesicles (GSVs) with the plasma membrane. In basal fat cells, insulin-responsive GSVs compartmentalize 50–80% of the total Glut4 pool with the rest of the transporter being present in endosomes, trans-Golgi network, and in nonspecialized intracellular transport vesicles (1). The mechanisms that control the distribution of Glut4 among different intracellular compartments are currently unknown. It is believed, however, that phosphoinositides and phosphoinositide kinases play a key role in the regulation of membrane traffic (2, 3). In particular, phosphatidylinositol 4-phosphate (PI4P), which has been considered mainly as a precursor for polyphosphorylated derivatives of phosphatidylinositol, is now emerging as an important regulator of intracellular trafficking in its own right (4, 5, 6).

Early results have demonstrated the presence of phosphatidylinositol 4-kinase (PI4K) in Glut4 vesicles from fat (7) and skeletal muscle (8) cells. However, Glut4 vesicles are not homogeneous and represent a mixture of at least two vesicular populations. These vesicular populations are different in size, protein composition, and functional properties (9, 10). A convenient marker, which is specifically present in only one vesicular population, is cellugyrin, a four transmembrane protein representing a ubiquitous analog of the major synaptic vesicle protein, synaptogyrin (11, 12). Cellugyrin-negative Glut4 vesicles, or GSVs, are enriched in Glut4 and insulin-regulated aminopeptidase and are translocated to the plasma membrane in response to insulin stimulation. Cellugyrin-positive vesicles are not enriched in Glut4 or insulin-regulated aminopeptidase, are not translocated to the plasma membrane, and may thus represent intracellular transport vesicles en route to GSVs. In agreement with this idea, further analysis has suggested that cellugyrin-positive vesicles represent ubiquitous intracellular transport vesicles present in all cell types tested (13). Because immunoadsorption with anti-Glut4 antibodies (7, 8) results in the isolation of both vesicular populations, it is not clear which compartment contains PI4K activity.

The molecular identity of PI4K associated with Glut4 vesicles has not been characterized either. At present, four different PI4Ks have been cloned in mammalian cells: PI4K type II (PI4KII) {alpha} and ß and PI4K type III {alpha} and ß (14). These enzymes are distinguished by differences in their enzymatic properties and by sensitivity to inhibitors. In particular, both type II kinases are resistant to wortmannin and are inhibited by adenosine, whereas both type III kinases are resistant to adenosine and sensitive to wortmannin (15).

Here, we report that PI4KII{alpha} is responsible for most of the PI4K activity associated with Glut4 vesicles. Virtually all this activity is associated with cellugyrin-positive vesicles and is not detectable in the GSVs. We have also determined that the unique N terminus of the enzyme is required for targeting of PI4KII{alpha} to cellugyrin-positive vesicles. Knockdown of PI4KII{alpha} with the help of short hairpin RNA (shRNA) does not affect the sedimentational distribution of cellugyrin-positive vesicles, suggesting that the biogenesis of the latter compartment does not involve PI4KII{alpha}.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
3T3-L1 adipocytes were homogenized and fractionated by centrifugation at 16,000 x g into the pellet and the supernatant. Small membrane vesicles (including both cellugyrin-positive vesicles and cellugyrin-negative GSVs) recovered in the supernatant of this centrifugation were purified further by immunoadsorption with 1F8 or with anticellugyrin beads (Fig. 1AGo). Material retained on the beads was subsequently eluted with 1% Triton X-100 and, then, with sodium dodecyl sulfate (SDS)-containing Laemmli sample buffer (16). Note, that Triton solubilizes all the vesicular proteins with the exception of those that directly interact with immobilized antibodies. The latter proteins are eluted only with SDS-containing buffers, such as Laemmli sample buffer. Immunoadsorption with 1F8 beads results in the isolation of virtually all Glut4-containing vesicles. After immunoadsorption with anticellugyrin beads, some Glut4 vesicles stay in the postadsorptive supernatant [unbound material (UB)], indicating that a population of Glut4 vesicles does not contain cellugyrin. PI4K activity and PI4KII{alpha} were present in the eluates from both 1F8 and anticellugyrin beads (Fig. 1Go, A and B), whereas PI4KIIß was not detectable in either fraction (data not shown).


Figure 1
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  		Fig. 1. PI4KII{alpha} and PI4K Activity Are Present in the Total Fraction of Glut4-Containing Vesicles and in Cellugyrin (Cg)-Containing Vesicles

3T3-L1 adipocytes were homogenized in PBS and subjected to centrifugation at 16,000 x g for 20 min. The resulting supernatant (600 µg in 200 µl) was incubated with 1F8 beads, anticellugyrin beads (Cg beads), and nonspecific IgG beads overnight. Unbound material was collected, and the beads were washed and subsequently eluted with 100 µl of 1% Triton X-100 in PBS and then with 100 µl of nonreducing Laemmli sample buffer. A, UB (30 µl), Triton X-100 eluate (TX, 50 µl), and SDS eluate (SDS, 50 µl) were analyzed by Western blotting. B, PI4K activity was measured in Triton X-100 eluates.

 
To specifically isolate cellugyrin-negative GSVs, we first removed cellugyrin-positive Glut4-vesicles by immunoadsorption with anticellugyrin beads, collected the postadsorptive supernatant (UB) and immunoisolated cellugyrin-negative Glut4 vesicles from this material using 1F8 beads. Figure 2Go shows that PI4KII{alpha} is present in cellugyrin-positive vesicles and is absent from cellugyrin-negative Glut4 vesicles. PI4KIIß is not associated with either compartment.


Figure 2
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  		Fig. 2. PI4KII{alpha} Is Specifically Associated with Cellugyrin (Cg)-Positive Vesicles and Is Absent from Cellugyrin-Negative GSVs

Immunoadsorption was sequentially performed with anticellugyrin beads and then with 1F8 beads as described in the legend to Fig. 1Go. UB, Triton X-100 eluate (TX), and SDS eluate (SDS) were analyzed by Western blotting. Nonspecific signal from the IgG heavy chain in SDS eluate is boxed.

 
To confirm this result, we separated cellugyrin-negative and cellugyrin-positive vesicles by centrifugation in a sucrose gradient, because cellugyrin-negative GSVs have a higher sedimentation coefficient than cellugyrin-positive vesicles. Figure 3Go demonstrates that, upon sucrose gradient centrifugation, the peak of PI4KII{alpha}-containing vesicles overlaps with the peak of cellugyrin-containing vesicles and not with the peak of cellugyrin-negative GSVs. PI4KIIß is present either in the cytosol in a free form or is associated with rapidly sedimenting membranes (see below) and, in agreement with previously shown data, is not present in either cellugyrin-positive or -negative Glut4 vesicles.


Figure 3
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  		Fig. 3. The Peak of PI4KII{alpha} Colocalizes with the Peak of Cellugyrin (Cg)-Positive Vesicles upon Sucrose Gradient Centrifugation

3T3-L1 adipocytes were homogenized in PBS and centrifuged at 16,000 x g for 20 min. The resulting supernatant (400 µg) was fractionated in a 10–30% continuous sucrose gradient for 55 min at 48,000 rpm in a Beckman SW-55 rotor at 4 C. The gradient was separated into 23 fractions, which were analyzed by Western blotting. Pel., Pellet.

 
With the help of double immunofluorescence staining, we found that the intracellular distribution of PI4KII{alpha} significantly overlaps with that of cellugyrin (Fig. 4Go). In addition, neither PI4KII{alpha} nor total PI4K activity is translocated to the plasma membrane in response to insulin stimulation (Fig. 5Go; see also Ref. 7). This result also supports the notion that PI4KII{alpha} is present not in insulin-responsive GSVs, but rather in cellugyrin-positive Glut4 vesicles that maintain intracellular localization both in the absence and in the presence of insulin (9, 10). Finally, we found that the expression levels of PI4KII{alpha} in differentiating 3T3-L1 cells are stable, which is more consistent with the expression pattern of cellugyrin, rather than Glut4 (Fig. 6Go).


Figure 4
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  		Fig. 4. Colocalization of PI4KII{alpha} with Cellugyrin

3T3-L1 adipocytes stably expressing cellugyrin-EGFP were serum starved in DMEM for 2 h, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and immunostained with a polyclonal primary antibody against PI4KII{alpha} and a Cy3-labeled secondary antibody. Staining with nonspecific IgG and the same secondary antibody did not produce any detectable signal and is not shown.

 

Figure 5
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  		Fig. 5. Insulin Does Not Have a Major Effect on the Intracellular Localization of PI4KII{alpha}

3T3-L1 adipocytes were incubated in serum-free DMEM for 2 h and treated with (+) or without (–) 100 nM insulin for 15 min at 37 C. The insulin action was stopped by the addition of KCN, and fat cells were fractionated into PM, HM, and LM fractions as described in Materials and Methods. A, Each fraction (50 µg) was analyzed by Western blotting. B, PI4K activity was measured in PM and LM fractions (50 µg each).

 

Figure 6
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  		Fig. 6. The Expression Level of PI4KII{alpha} Remains Stable during Differentiation of 3T3-L1 Adipocytes

3T3-L1 preadipocytes were cultured and differentiated as described in Materials and Methods. Cell extracts were prepared on each day of differentiation and analyzed by Western blotting.

 
Thus, we found that PI4KII{alpha} is virtually undetectable in cellugyrin-negative GSVs but is considerably enriched in cellugyrin-positive population of Glut4-vesicles. To determine whether or not type III PI4K can contribute to the total PI4K activity in this compartment, we measured this activity in the presence of wortmannin and adenosine. In an agreement with previously published results (7, 8), we found that PI4K activity in cellugyrin-positive vesicles is resistant to wortmannin and is virtually completely inhibited by adenosine (Fig. 7Go). In parallel experiments, we determined that wortmannin completely inhibited insulin-activated glucose transport and Glut4 translocation in 3T3-L1 adipocytes (data not shown). Taken together, these results suggest that PI4KII{alpha} is the major or the only PI4K in this compartment.


Figure 7
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  		Fig. 7. PI4K Activity Associated with Cellugyrin-Positive Vesicles Is Inhibited by Adenosine, But Not by Wortmannin

A, Cellugyrin-positive vesicles were immunoisolated from 3T3-L1 adipocytes and subjected to PI4K assay as described in Materials and Methods in the absence (Control) and in the presence of 500 nM wortmannin (Wort) or 200 µM adenosine (Adenosine). B, PI4K activity in cellugyrin-positive vesicles was measured in the presence of different concentrations of adenosine.

 
We believe that cellugyrin-positive vesicles represent the first biochemically homogeneous membrane compartment that specifically contains PI4KII{alpha} and not a closely related isoform, PI4KIIß. This raises the question of what is the nature of the targeting signal that is responsible for the presence of PI4KII{alpha} in these vesicles. Interestingly, a recent report shows that PI4KII{alpha} is also associated with synaptic vesicles in the brain (17), a compartment that may be evolutionarily related to ubiquitous cellugyrin-containing vesicles (see Discussion). However, because PI4KIIß is undetectable in neurons (17), it remains unclear whether PI4KII{alpha} is targeted to synaptic vesicles by a specific sequence or is present there simply by virtue of being the only isoform of PI4K expressed in this cell type.

To study targeting of PI4KII{alpha} and PI4KIIß, we transiently expressed these enzymes in human embryonic kidney (HEK) 293 cells. Cells were homogenized and centrifuged at 27,000 x g to separate the fraction of small vesicles from heavy membranes. The supernatant of the 27,000 x g centrifugation was further fractionated in a linear sucrose velocity gradient. Figure 8Go, A and B, shows that PI4KII{alpha} is targeted to cellugyrin-positive vesicles, whereas all PI4KIIß in the 27,000 x g supernatant cosediments with free soluble proteins. This result strongly suggests that localization of PI4KII{alpha} in cellugyrin-positive vesicles requires a specific targeting sequence. A significant fraction of both PI4KII{alpha} and PI4KIIß is recovered in the pellet of the 27,000 x g centrifugation (Fig. 8GGo), indicating that these enzymes are also associated with heavy membrane structures, such as endosomes and/or Golgi apparatus (18, 19).


Figure 8
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  		Fig. 8. Targeting of PI4K{alpha} to Cellugyrin (Cg)-Positive Vesicles Requires the N Terminus of the Enzyme

A–F, HEK 293 cells were transiently transfected with myc-tagged wild-type PI4KII{alpha}, wild-type PI4KIIß, and truncated mutants of PI4KII{alpha}. Cells were fractionated as described in Materials and Methods, and the supernatant of the 27,000 x g centrifugation (400 µg) was fractionated in a 10–30% continuous sucrose gradient for 1.5 h at 48,000 rpm in a Beckman SW-55 rotor at 4 C. The gradient was separated into 23–25 fractions, which were analyzed by Western blotting with a monoclonal antibody against the myc epitope and with a polyclonal antibody against cellugyrin. G, Total expression levels of the constructs in PNS and in the pellet of the 27,000 x g centrifugation. Panels show Western blot with the anti-myc antibody.

 
PI4KII{alpha} and PI4KIIß demonstrate a high degree of similarity with most of the differences localized in the N termini of the enzymes (18, 20). We have created four mutants of PI4KII{alpha} with the truncated N terminus [amino acids (a.a.) 94–479, 104–479, 125–479, and 147–479] and expressed them in HEK 293 cells. As is shown in Fig. 8Go, C–F, truncation of the N terminus leads to a gradual decrease in the efficiency of targeting of PI4KII{alpha} to cellugyrin-positive vesicles, so that the last mutant (a.a. 147–479) is distributed to the fraction of free soluble proteins similar to PI4KIIß. Because all these mutants are present in the pellet of the 27,000 x g centrifugation (Fig. 8GGo), we believe that their general membrane targeting [mediated by palmitoylation of a cystein-rich motif in the middle of the protein (15)] is intact, and that the N terminus of PI4KII{alpha} may specifically be required for targeting of the enzyme to small cellugyrin-positive vesicles.

The presence of PI4KII{alpha} in cellugyrin-positive vesicles suggests that this enzyme may be involved in the biogenesis of this compartment. To test this hypothesis, we have knocked down PI4KII{alpha} in HEK 293 cells using the RNA interference approach (5). We were able to achieve a significant decrease in the total levels of PI4KII{alpha} (Fig. 9Go A). In particular, the presence of the enzyme in cellugyrin-positive vesicles becomes virtually undetectable (Fig. 9BGo). This, however, does not have any effect on the sedimentational distribution of cellugyrin, suggesting that PI4KII{alpha} may not be involved in the formation or trafficking of cellugyrin-positive vesicles.


Figure 9
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  		Fig. 9. Knockdown of PI4K{alpha} Does Not Affect Cellugyrin (Cg)-Positive Vesicles

PI4KII{alpha} in HEK 293 cells was knocked down as described in Materials and Methods. A, Total cell lysate was analyzed by Western blotting. B, Supernatant of the 27,000 x g centrifugation (400 µg) was fractionated in a 10–30% continuous sucrose gradient for 1.5 h at 48,000 rpm in a Beckman SW-55 rotor at 4 C. The gradient was separated into 23 fractions, which were analyzed by Western blotting with polyclonal antibodies against PI4KII{alpha} and cellugyrin. EV, Empty vector.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Previous studies with overexpressed tagged proteins have demonstrated that PI4KII{alpha} and PI4KIIß have overlapping but still distinct intracellular localization, although the biochemical nature of the PI4KII-containing compartments is still a matter of debate (18, 19). Here, we studied the intracellular localization of endogenous enzymes using predominantly a biochemical approach and identified an individual membrane compartment (called cellugyrin-positive transport vesicles) that specifically contains PI4KII{alpha} but not other isoforms of PI4K. This suggests that different isoforms of the enzyme play nonredundant functions in the cell. We show, furthermore, that targeting of PI4KII{alpha} to cellugyrin-positive vesicles requires the unique N terminus of the enzyme.

What is the biological nature of cellugyrin-positive vesicles? Originally, this compartment was identified as a subpopulation of Glut4 vesicles (9). However, based on the protein composition of cellugyrin vesicles and their intracellular localization and rapid accumulation of recycling proteins, we have suggested that cellugyrin-containing vesicles may mediate protein transport from sorting endosomes to the endocytic recycling compartment and/or to trans-Golgi network and may thus represent a ubiquitous type of an intracellular vesicular carrier (9, 10). Indeed, further analysis has demonstrated that uniform cellugyrin-containing vesicles are present in all cell types studied (13). Interestingly, overexpression of cellugyrin increases the number of vesicles, suggesting that this protein may play a key role in vesicle biogenesis (21).

An important concern with the in vitro analysis of small membrane compartments is that they may represent an artificial product of large membrane structures, such as endosomes, that may be fragmented into smaller vesicles upon cell homogenization. However, at least two lines of evidence strongly suggest that cellugyrin-containing vesicles do not represent an artifact of cell homogenization, but exist in vivo. First, special control experiments demonstrate that homogenization does not affect the yield and the size of these vesicles (10). Second, we have established an in vitro budding assay for cellugyrin-containing vesicles and demonstrated that these vesicles are formed in vitro from heavy membranes, presumably endosomes, in a cytosol-, ATP-, time-, and temperature-dependent fashion (22). Thus, because cellugyrin-containing vesicles are present in extracts of nonhomogenized cells and are formed in vitro in a physiological process, we believe that these vesicles do not represent an artifact of homogenization, but exist in the living cell.

Recently, Guo et al. (17) have found that PI4KII{alpha} is responsible for PI4K activity in synaptic vesicles. Interestingly, there are several parallels between specialized synaptic vesicles in neurons and ubiquitous cellugyrin-containing vesicles. In particular, these types of vesicles have identical sedimentation coefficients (13) and contain secretory carrier membrane proteins, synapto/cellubrevin, and synapto/cellugyrin as their major component proteins. According to one hypothesis, synaptic vesicles may develop from ubiquitous microvesicles in the process of neuronal differentiation by substitution of the ubiquitously expressed protein isoforms with their neuronal counterparts. Indeed, differentiating mouse brain at embryonic d 16 does not yet express synaptic proteins and has only cellugyrin-containing microvesicles. With differentiation, expression of synaptic proteins, such as synaptobrevin and synaptogyrin, is increased whereas expression of cellubrevin and cellugyrin gradually terminates (Dr. J. K. Blusztajn, Boston University School of Medicine, personal communication) which may lead to transformation of ubiquitous transport vesicles into highly specialized synaptic vesicles. The fact that PI4KII{alpha} is present in both types of vesicles suggests that it may be involved in some basic nonspecialized aspects of the vesicular traffic. Knockdown experiments (Fig. 9Go) demonstrate that the direct involvement of PI4KII{alpha} in the biogenesis of this vesicular compartment is unlikely. One hypothesis is that PI4KII{alpha} may use small vesicular carriers as vessels to move from one subcellular compartment to another, i.e. from the plasma membrane to sorting endosomes or from sorting endosomes to recycling endosomes and/or trans-Golgi network. This may give the cell an opportunity to balance the endocytic and the exocytic PI4P-dependent membrane flow.

Recently, Salazar et al. (23) showed that PI4KII{alpha} represented a component of AP3-derived vesicles. Although this result does not exclude a possibility that PI4KII{alpha} may be present in other vesicular types as well, it indicates that ubiquitous cellugyrin-positive vesicles may be formed in an activator protein (AP)3-dependent fashion. Furthermore, PI4P is known to recruit the adaptor complex AP1 to the donor membranes (5). Thus, if cellugyrin-positive vesicles are indeed formed via the AP3-mediated mechanism, our results suggest that recruitment of AP3 to the membranes may be PI4P independent. This hypothesis deserves further investigation.

Recently, Mora et al. (24) using confocal immunofluorescence microscopy found that PI4KIIIß and its calcium-sensing partner neuronal calcium sensor-1 show a significant colocalization with Glut4 in 3T3-L1 adipocytes. Because PI4K type III is not present in small Glut4-vesicles (Refs. 7 and 8 and present paper), we suggest that PI4KIIIß may colocalize with Glut4 in some other intracellular compartment(s), e.g. in cis/medial Golgi where PI4KIIIß is abundant (25).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Dexamethasone, 3-isobutyl-1-methylxanthine, insulin, fetal bovine serum, and donkey serum were purchased from Sigma (St. Louis, MO). Aprotinin, leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride were obtained from American Bioanalytical (Natick, MA). Calf serum, Lipofectamine 2000, and DMEM were purchased from Invitrogen (Carlsbad, CA).

Antibodies
A monoclonal anti-Glut4 antibody 1F8 (26) and a rabbit polyclonal antibody against cellugyrin (22) were described previously. Rabbit polyclonal antibodies against PI4KII{alpha} and PI4KIIß were kind gifts from Dr. Pietro De Camilli (Yale University School of Medicine, New Haven, CT). The monoclonal antibody against the myc epitope was purchased from Cell Signaling Technology (Beverly, MA).

Engineering of PI4KII{alpha} and PI4KIIß Constructs
The cDNA (IMAGE clone indentification no. 2905670) encoding human PI4KII{alpha} and the cDNA (IMAGE clone identification no. 6335661) encoding mouse PI4KIIß were purchased from American Type Culture Collection (Manassas, VA) and Invitrogen, respectively. PI4KII{alpha} and PI4KIIß open reading frames were amplified by PCR and subcloned into pCDNA3.1 His B vector (Invitrogen) via HindIII and EcoRI sites. Myc tags were introduced to the 5'-ends of the open reading frames via sense primers. For better expression in mammalian cells, Kozak sequences were included in the sense primers. Four mutants of PI4KII{alpha} with the truncated N terminus (a.a. 94–479, 104–479, 125–479, 147–479) were obtained with the help of PCR.

The sense primers for PCR were:

PI4KII{alpha} full length,

5'-CGCAAGCTTACCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGGACGAGACGAGCCC-3',

PI4KII{alpha} 94–479,

5'-CGCAAGCTTACCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTG GCCGCTCAGGCCCAC-3',

PI4KII{alpha} 104–479,

5'-CGCAAGCTTACCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGTTCCCGGAGGATCCTG-3',

PI4KII{alpha} 125–479,

5'-CGCAAGCTTACCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGATCTTTCCCGAGCGCATC-3',

PI4KII{alpha} 147–479,

5'-CGCAAGCTTACCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGATCATTGCTGTCTTCAAACCC-3',

PI4KIIß full length,

5'-GGCAAGCTTACCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGGCGGAGGCCTGCGAG-3'

The antisense primer for PI4KII{alpha} constructs was 5'-GCGAATTCCTACCACCATGAAAAGAAGGG-3'.

The antisense primer for PI4KIIß construct was 5'-GCGAATTCCTACCAGGAGGAGAAGAACGG-3'.

RNA Interference Constructs
PI4KII{alpha} shRNA construct was created using the sequence between nucleotides 888 and 908 (GenBank accession no. NM_18425) (5) and the protocol for the design and creation of PCR-based shRNA available at http://katahdin.cshl.org:9331/RNAi_web/scripts/main2.pl. Resulting shRNA (5'-TGCTGTTGACAGTGAGCGCTGAAGCAGAACCTCTTCCTGATAGTGAAGCCACAGATGTATCAGGAAGAGGTTCTGCTTCAATGCCTACTGCCTCGGA-3') was amplified by PCR using the following primers:

5'-CAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCG-3' and 5'-CTAAAGTAGCCCCTTGAATTCCGAGGCAGTAGGCA-3'. The PCR product was digested with XhoI and EcoRI and cloned into the pSM2 vector (Open Biosystems, Huntsville, AL).

Cell Culture
Murine 3T3-L1 preadipocytes were cultured, differentiated, and maintained as described previously (22). Briefly, cells were grown in DMEM supplemented with 10% calf serum until confluence. The cells were transferred 2 d later to differentiation medium (DMEM containing 10% fetal bovine serum, 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, and 1.7 µM insulin). After 48 h, the differentiation medium was replaced with maintenance medium (DMEM supplemented with 10% fetal bovine serum). The maintenance medium was changed every 48 h. Before each experiment, cells were starved in serum-free media for 2 h.

HEK 293 FT cells were cultured in DMEM supplemented with 10% fetal bovine serum plus 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Transient transfections were performed according to the manufacturer’s instructions, and cells were used 48 h after transfection.

To establish a stable PI4KII{alpha} knockout cell line, the pSM2 vector with and without PI4KII{alpha} shRNA was transfected into HEK293 cells using lipofectamine 2000, and 7.5 µg/ml puromycin was added to the media 1 d after transfection. Selection was carried out for 2 wk, and the pooled population of cells was used.

Subcellular Fractionation
3T3-L1 adipocytes (d 8 after initiation of differentiation) were washed twice with 37 C DMEM and then serum starved for 2 h. Cells were treated with either 100 nM insulin or with carrier (5 mM HCl) in DMEM for 15 min at 37 C, and KCN was added to the cells at a final concentration of 0.2 mM. Cells were washed three times and were homogenized in PBS with the protease inhibitor cocktail (1 µM aprotinin, 1 µM pepstatin, 1 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride) using a ball-bearing homogenizer (Isobiotec, Heidelberg, Germany) with a 12-µm clearance. The homogenate was fractionated by differential centrifugation into plasma membrane (PM), heavy microsomes (HM), and light microsomes (LM) as previously described (27, 28). Alternatively, the homogenate was centrifuged at 16,000 x g for 20 min to pellet PM and other heavy membranes, and supernatant of this centrifugation which contained HM and LM, as well as free soluble proteins, was used for further experiments.

HEK-293 FT cells were homogenized using the ball-bearing homogenizer as described above. The homogenate was centrifuged at 500 x g for 5 min to generate the postnuclear supernatant (PNS). The PNS was then centrifuged at 27,000 x g for 35 min, and the resulting supernatant was analyzed by sucrose gradient centrifugation.

Sucrose Gradient Centrifugation
Samples (0.2 ml) were loaded onto a 4.6-ml continuous 10–30% (wt/wt) sucrose gradient and centrifuged in a Beckman SW-55 Ti rotor at 48,000 rpm for indicated periods of time. Each gradient was fractionated into 23–25 fractions starting from the bottom of the tube.

Immunoadsorption
Affinity-purified 1F8 antibody and anticellugyrin antibody, as well as nonspecific mouse or rabbit IgG (Sigma), were each coupled to Dynal magnetic beads (Dynal Biotech, Carlsbad, CA) according to the manufacturer’s instructions. Usually, 2 µg of each antibody was taken for 30 µl of the beads. Before use, the antibody-coupled beads were blocked with 1% BSA in PBS for 30 min at 4 C, followed by a wash with cold PBS. The 16,000 x g supernatants (600 µg) from 3T3-L1 adipocytes were incubated with 30 µl 1F8 beads, anticellugyrin beads, or nonspecific IgG beads overnight at 4 C with gentle shaking. The beads were washed three times with PBS and then eluted with 1% Triton X-100 in PBS for 1 h at 4 C. After Triton elution, the beads were washed three times with PBS and eluted with nonreducing Laemmli sample buffer.

Gel Electrophoresis and Western Blotting
Proteins were separated in SDS-polyacrylamide gels according to Laemmli (16), but without reducing agents, and transferred to an Immobilon-P membrane (Millipore Corp., Bedford, MA) in 25 mM Tris, 192 mM glycine. After transfer, the membrane was blocked with 10% nonfat milk in PBS with 0.5% Tween 20 for 1 h at 37 C. The blots were probed with specific antibodies and horseradish peroxidase-conjugated secondary antibodies (Sigma) and detected with an enhanced chemiluminescence substrate kit (PerkinElmer Life Sciences, Boston, MA) using a Kodak Image Station 440CF (Eastman Kodak, Rochester, NY). The signals were quantified using the Kodak 1D image analysis software.

Immunofluorescence
3T3-L1 adipocytes stably expressing cellugyrin-enhanced green fluorescent protein (EGFP) were grown and differentiated as described above. On d 7 of differentiation, cells were trypsinized and replated on coverslips in 12-well plates. The following day, cells were serum starved in DMEM for 2 h, washed once with PBS, and then fixed with 4% formaldehyde in PBS for 30 min at room temperature. Cells were washed twice with PBS and permeabilized with PBS containing 0.2% Triton X-100 for 5 min at room temperature. The cells were then rinsed three times with PBS followed by blocking for 1 h at room temperature in PBS containing 5% donkey serum and 5% BSA (blocking solution). The cells were then incubated for 1 h with a polyclonal antibody against PI4KII{alpha} at a dilution of 1:1000 in the blocking solution at room temperature. Cells were rinsed six times with PBS, covered with aluminum foil, and incubated for another hour at room temperature with Cy3-labeled antirabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:1000 in blocking solution. The cells were washed six times with PBS and mounted on slides with the help of SlowFade Antifade Kit (Molecular Probes, Eugene, OR). Stained cells were visualized with the help of the Axiovert 200M microscope (Carl Zeiss, Thornwood, NY). Pictures were taken using the Axiovision 3.0 software (Carl Zeiss).

PI4K Assay
Samples (5 µl) were mixed with 50 µg of phosphatidyl inositol (Avanti Polar Lipids, Alabaster, AL) and Triton X-100 (0.4% final concentration) in PI4K buffer (30 mM HEPES, pH 7.4; 100 mM NaCl; 2 mM MgCl2; 0.5 mg/ml BSA) in a total volume of 50 µl. The reaction was initiated by the addition of 40 µM ATP (10 µCi of [{gamma}-32P]ATP per assay) and terminated after a 10-min incubation at room temperature by the addition of 100 µl of 1 M HCl. Lipids were extracted with 160 µl of the chloroform-methanol mixture (1:1) by vortexing, and the samples were microfuged for 5 min. The lower phase was extracted with 80 µl methanol/100 mM HCl with 2 mM EDTA (1:1), and the samples were centrifuged again in the same regimen. The lower phase of the second extraction was collected and subjected to thin layer chromatography on potassium oxalate-pretreated silica gel 60 plates (Whatman, Florham Park, NJ) using chloroform-acetone-methanol-acetic acid-water (60:23:20:18:12) as a mobile phase. Commercial phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol-4,5-biphosphate (Avanti Polar Lipids) were used as standards. They were visualized with iodine vapor and marked with fluorescent labels. 32P-labeled phosphoinositides were detected by autoradiography using the InstantImager (PerkinElmer).


   ACKNOWLEDGMENTS
 
We thank Dr. Lin V. Li (Boston University School of Medicine) for 3T3-L1 cell line stably expressing cellugyrin-EGFP and to Dr. Pietro De Camilli (Yale University School of Medicine) for his generous gift of antibodies.


   FOOTNOTES
 
This work was supported by research grants DK52057 and DK56736 from the National Institutes of Health (to K.V.K.).

The authors have nothing to disclose.

First Published Online June 13, 2006

Abbreviations: a.a., Amino acids; AP, adaptor protein; EGFP, enhanced green fluorescent protein; Glut4, glucose transporter 4; GSV, Glut4 storage vesicle; HEK, human embryonic kidney; HM, heavy microsome; LM, light microsome; PI4K, phosphatidylinositol 4-kinase; PI4KII, type II PI4K; PI4P, phosphatidylinositol 4-phosphate; PM, plasma membrane; PNS, postnuclear supernatant; SDS, sodium dodecyl sulfate; shRNA, short hairpin RNA; UB, unbound material.

Received for publication May 8, 2006. Accepted for publication June 8, 2006.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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