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Originally published In Press as doi:10.1074/jbc.M601590200 on May 22, 2006

J. Biol. Chem., Vol. 281, Issue 31, 22261-22274, August 4, 2006
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Mechanism of Assembly of G Protein beta{gamma} Subunits by Protein Kinase CK2-phosphorylated Phosducin-like Protein and the Cytosolic Chaperonin Complex*

Georgi L. Lukov, Christine M. Baker, Paul J. Ludtke, Ting Hu, Michael D. Carter, Ryan A. Hackett, Craig D. Thulin, and Barry M. Willardson1

From the Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602

Received for publication, February 21, 2006 , and in revised form, April 28, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein beta{gamma} subunit complex (Gbeta{gamma}) that has been recently shown to catalyze the formation of the Gbeta{gamma} dimer from its nascent polypeptides. Phosphorylation of PhLP at one or more of three consecutive serines (Ser-18, Ser-19, and Ser-20) is necessary for Gbeta{gamma} dimer formation and is believed to be mediated by the protein kinase CK2. Moreover, several lines of evidence suggest that the cytosolic chaperonin complex (CCT) may work in concert with PhLP in the Gbeta{gamma}-assembly process. The results reported here delineate a mechanism for Gbeta{gamma} assembly in which a stable ternary complex is formed between PhLP, the nascent Gbeta subunit, and CCT that does not include G{gamma}. PhLP phosphorylation permits the release of a PhLP·Gbeta intermediate from CCT, allowing G{gamma} to associate with Gbeta in this intermediate complex. Subsequent interaction of Gbeta{gamma} with membranes releases PhLP for another round of assembly.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Eukaryotic cells employ heterotrimeric G proteins to transduce a wide variety of hormonal, neuronal, and sensory signals that control numerous physiological processes. As a result, malfunctions in G protein pathways contribute to many diseases (1-3), and therapeutic agents targeting G protein-coupled receptors represent the single largest class of current pharmaceuticals (4). There are three fundamental steps in the propagation of a G protein-mediated signal. First, a ligand binds a receptor, resulting in a change in the packing of the seven transmembrane {alpha}-helices found in all G protein-coupled receptors. Second, the activated receptor catalyzes exchange of GDP for GTP on the {alpha} subunit of a heterotrimeric G protein (G{alpha})2 on the intracellular surface of the receptor. GTP binding causes G{alpha} to dissociate from the G protein beta{gamma} subunit complex (Gbeta{gamma}). Third, the G{alpha}·GTP and Gbeta{gamma} complexes control the activity of effector enzymes and ion channels that regulate the intracellular concentration of second messengers (cyclic nucleotides, inositol phosphates, and Ca2+) and the plasma membrane electrical potential (mainly via K+ channels). Changes in these properties in turn orchestrate the cellular response to the stimulus (5).

Phosducin-like protein (PhLP) is a member of the phosducin gene family (6-8) that is believed to participate in G protein signaling by virtue of its ability to bind the Gbeta{gamma} dimer with high affinity (9-11). Many in vitro and overexpression experiments have shown that PhLP binding to Gbeta{gamma} blocks its ability to interact with G{alpha} or effectors (9, 10, 12-14). From these experiments, it was suggested that the physiological role of PhLP was to down-regulate G protein signaling by sequestering Gbeta{gamma}. However, the results of several recent studies have seriously challenged this model. Specifically, disruption of the PhLP1 gene in the chestnut blight fungus Cryphonectria parasitica (15) and in the soil amoeba Dictyostelium discoideum (7) yielded the same phenotype as the disruption of the Gbeta gene. Moreover, PhLP deletion blocked G protein signaling in Dictyostelium (7). In another study, the duration of opiate desensitization was prolonged in mice in which PhLP expression in the brain was inhibited by antisense oligonucleotide treatment (16). All of these observations are the exact opposite of what would be predicted by the Gbeta{gamma} sequestration model.

Insight into an alternative function of PhLP has come from the observation that PhLP interacts with the cytosolic chaperonin containing tailess complex polypeptide 1 (CCT), an essential molecular chaperone that mediates the folding of actin, tubulin, and other proteins into their native structures (17). PhLP was shown to interact with CCT as a regulator and not as a folding substrate. In addition, the cryoelectron microscopic structure of the PhLP·CCT complex (18) shows that PhLP binds CCT at the top of the CCT apical domains positioned above the folding cavity in a manner analogous to prefoldin, a CCT co-chaperone that binds nascent actin polypeptide chains and delivers them to CCT for folding (19). Coupling these observations with the fact that yeast Gbeta (20) and other proteins with seven beta-propeller structures similar to Gbeta (21-23) interact with CCT suggests that PhLP might function as a co-chaperone in the folding of Gbeta. Indeed, recent findings show that PhLP does act as an essential chaperone for assembly of the Gbeta{gamma} dimer, demonstrating that the physiological function of PhLP is not to down-regulate G protein signaling by sequestering Gbeta{gamma} but to support G protein signaling by catalyzing Gbeta{gamma} dimer formation (24). However, the role of CCT in this process remains unclear (24, 25).

Phosphorylation of PhLP by protein kinase CK2 (CK2) plays an important role in PhLP function. A major site of CK2 phosphorylation occurs within a sequence of three consecutive serines (residues 18-20) near the N terminus (14). Phosphorylation of serines 18-20 was required for PhLP-mediated Gbeta{gamma} assembly, for when these residues were substituted with alanine, PhLP was unable to catalyze Gbeta{gamma} dimer formation (24). The mechanism by which phosphorylation at these sites enhances Gbeta{gamma} dimer formation is not known; therefore, the effects of CK2 phosphorylation on PhLP function were investigated. The results of these studies provide evidence for a mechanism of PhLP-mediated Gbeta{gamma} assembly that involves the formation of a ternary complex between PhLP, the nascent Gbeta polypeptide, and CCT. PhLP phosphorylation is required for the release of PhLP·Gbeta from the CCT complex and the subsequent association of G{gamma} with Gbeta to form the Gbeta{gamma} dimer.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—HEK-293 and CHO cells were cultured in Dulbecco's modified Eagle's medium/F-12 (50/50 mix) growth media with L-glutamine and 15 mM HEPES, supplemented with 10% fetal bovine serum (HyClone). The cells were subcultured regularly to maintain active growth but were not used beyond 20-25 passages.

Preparation of cDNA Constructs—Wild-type human PhLP, PhLP {Delta}1-75, and Pdc with 3' c-myc and His6 tags in the pcDNA3.1/myc-His B vector (Invitrogen) were prepared as described (24). Serine-to-alanine variants of human PhLP at positions 18, 19, 20, 25, and 296 were constructed in the pcDNA3.1/myc-His B vector by employing a PCR-based strategy and utilizing unique endonuclease restriction sites near the substitution site as described (24). The constructs were then subcloned into the bacterial expression vector pET15b (Novagen) as described (26). The integrity of all constructs was confirmed by sequence analysis. The N-terminally hemagglutinin (HA)-tagged G{gamma}2, FLAG epitope-tagged Gbeta1 and untagged Gbeta1 cDNAs also in the pcDNA3.1 vector were obtained from the UMR cDNA Resource Center.

Protein Expression and Purification—Wild-type and CK2 phosphorylation site variants of human PhLP in the pET15b vector were transformed in Escherichia coli DE3 cells and were purified using nondenaturing Ni2+ affinity chromatography as described previously (11). The purified proteins were concentrated and exchanged into 20 mM HEPES, pH 7.2, 150 mM NaCl by ultrafiltration and were stored in 50% glycerol at -20 °C. Purified Gbeta1{gamma}1 and retinal rod outer segment membranes were prepared also as described previously (11). Protein concentrations were determined using Coomassie Plus protein assay reagent (Pierce), and the purity was determined to be ~95% by SDS-PAGE.

CK2 Phosphorylation of PhLP—Purified PhLP (50 µM) was phosphorylated by CK2 (10 units/µl, Calbiochem) in 20 mM HEPES, pH 7.5, 100 mM KCl, 20 mM NaCl, 5 mM MgCl2, 5 mM dithiothreitol, and 1 mM ATP for 1 h at 37°C. The phosphorylation was confirmed by SDS-PAGE using 10% gels.

Assay of PhLP Binding to CCT—The binding of CK2 phosphorylated or unphosphorylated PhLP and its CK2 phosphorylation site variants to CCT was measured by adding 5 nM purified PhLP to 5% rabbit reticulocyte lysate in phosphate-buffered saline containing 1.2% IGEPAL CA-630 (Sigma) and 0.6 mM phenylmethylsulfonyl fluoride in a total volume of 300 µl. Binding was allowed to proceed for 30 min at 23 °C after which the PhLP was immunoprecipitated by incubating for 30 min at 4 °C with 2.1 µg of anti-c-myc antibody (clone 9E10, Biomol) followed by the addition of 30 µl of a 50% slurry of Protein A/G Plus-agarose beads (Santa Cruz Biotechnology) and another 30-min incubation at 4 °C with constant mixing. The beads were washed with the phosphate-buffered saline/IGEPAL buffer, and proteins were solubilized with 20 µl of 2x SDS-PAGE sample buffer. 15 µl of each sample was resolved on a 10% SDS-PAGE gel and was immunoblotted with a 1:1000 dilution of rabbit polyclonal anti-CCT{epsilon} antiserum (18) followed by a 1:2000 dilution of a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Calbiochem). Immunoblots were developed with the ECL Plus chemiluminescence reagent (GE Healthcare) and visualized with a Storm 860 PhosphorImager (Amersham Biosciences). The band intensities were quantified using Image-QuaNT software (Amersham Biosciences).

Assay of PhLP Inhibition of Gt Binding to Rhodopsin—Light-induced binding of 0.2 µM 125I-labeled Gt{alpha} and 0.2 µM Gtbeta{gamma} to membranes containing 1 µM rhodopsin ± 2 µM CK2 phosphorylated or unphosphorylated PhLP was measured as described previously (11).

Mass Spectrometric Analyses—Tryptic peptides of CK2-phosphorylated PhLP were generated and analyzed as described previously (26). Briefly, molecular ions in the effluent from a C18 capillary chromatography that corresponded to the predicted masses of phosphopeptides from PhLP were fragmented by collision-induced dissociation in a Q-ToF mass spectrometer (LC/MS/MS). Fragmentation spectra were obtained using either automated or manual parent ion selection. Data were analyzed using BioAnalyst software (Applied Biosystems, Framingham, MA).

Electrophoretic Mobility Determinations—CHO cells were plated in 6-well plates so that they were 70-80% confluent the next day. The cells were then transfected with 1 µg of either wild-type PhLP-myc or one of the CK2 phosphorylation site variants using Lipofectamine Plus reagent according to the manufacturer's protocol. The cells were harvested 48 h later in 200 µl of immunoprecipitation buffer (24), and the PhLP-myc was immunoprecipitated from the lysate with 3 µg of anti-c-myc antibody and 30 µl of Protein A/G beads as described previously (17, 24). The final precipitate was solubilized in 40 µl of 2x SDS-PAGE sample buffer, and 10 µl of each sample was resolved on 10% SDS-PAGE gels. The gels were immunoblotted with a 1:1000 dilution of the anti-c-myc antibody and developed as described above.

Gbeta{gamma} Expression Measurements—HEK-293 cells were plated in 6-well plates so that they would be 70-80% confluent the following day. They were then co-transfected with 1 µg of each of the PhLP-myc, HA-G{gamma}2, and Gbeta1 cDNAs using Lipofectamine Plus reagent. In all experiments involving multiple transfections, the total amount of cDNA was held constant by adding empty vector. After 48 h, the cells were washed and solubilized in 200 µl of immunoprecipitation buffer. The Gbeta1{gamma}2 complexes were immunoprecipitated from 100 µl of the lysate with 1.5 µg of anti-HA (clone 3F10, Roche Applied Science) antibody as described previously (17, 24). The complexes were solubilized in 40 µl of 2x SDS sample buffer, and 10 µl was resolved on 10% Tris-glycine SDS-PAGE gels for Gbeta1, or 20 µl was resolved on 16.5% Tris-Tricine SDS-PAGE gels for G{gamma}2. For Gbeta detection, the gels were immunoblotted with a 1:2000 dilution of a Gbeta1 antibody as described (27). For HA-G{gamma}2, the membranes were blocked for 1 h in 2% bovine serum albumin in Tris-buffered saline, followed by incubation for 1 h in a 1:500 dilution of anti-HA antibody in the blocking buffer and then 1 h in a 1:2000 dilution of goat anti-rat horseradish peroxidase conjugated secondary antibody (Calbiochem). The immunoblots were developed as described above.


Figure 1
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  		FIGURE 1.
Effects of CK2 phosphorylation on PhLP binding to CCT and Gbeta{gamma}. A, the decrease in mobility of PhLP in SDS-PAGE upon CK2 phosphorylation is shown. PhLP was phosphorylated by CK2 in vitro (P-PhLP) and was analyzed on a 10% gel along with unphosphorylated PhLP. B, the effects of CK2 phosphorylation on the binding of PhLP to CCT and Gbeta{gamma} are shown. Binding was measured by immunoprecipitation of PhLP coupled with detection of the co-immunoprecipitating CCT{epsilon} or Gbeta by immunoblotting. A representative immunoblot is shown. The graph gives the average intensity ± S.E. of the CCT bands relative to the unphosphorylated sample from eight separate experiments. C, the effects of CK2 phosphorylation on the ability of PhLP to inhibit Gbeta1 {gamma}1-assisted binding of 125I-labeled Gt{alpha} to membranes containing light-activated rhodopsin were determined. The graph gives the average ± S.E. from three separate experiments.

 
Radiolabel Pulse-Chase Assay—The pulse-chase assay was performed and quantified as described previously (24). A similar protocol was used to measure the rate of release of nascent Gbeta from CCT. Six-well plates of HEK-293 cells were co-transfected with 1.0 µg of FLAG Gbeta1, HA-G{gamma}2 or PhLP-myc variants as indicated. After a 10-min pulse, the radiolabel was chased for the times indicated and the cells were harvested in 220 µl of immunoprecipitation buffer. The extract was divided into two 95-µl samples, and 2.5 µl of 1 µg/µl anti-CCT{epsilon} antibody (Serotec) was added to one sample and 3.0 µl of 1 µg/µl anti-FLAG antibody was added to the other sample. The immunoprecipitation and analysis of the radiolabeled proteins co-immunoprecipitating with CCT were carried out as described (24). The Gbeta1 band was clearly separated from the other radiolabeled bands, facilitating its quantification. The amount of Gbeta1 in the CCT immunoprecipitate was divided by that in the FLAG-Gbeta1 immunoprecipitate to determine the fraction of the total Gbeta1 bound to CCT. These values were expressed as a percentage of the 30-min time point to readily compare the rates of Gbeta dissociation from CCT. The data were fit to a first order dissociation rate equation using the KaleidaGraph graphics software to determine the dissociation rate constant k. From the k values, the half-life was calculated by the equation, t1/2 = ln 2/k.

Assay of Gbeta Binding to CCT—For CCT binding experiments involving Gbeta1 overexpression, HEK-293 cells were plated in 6-well plates and transfected with 1.0 µg of FLAG-Gbeta1, HA-G{gamma}2, Pdc, or PhLP cDNAs as indicated in Fig. 5 (A and C). Alternatively, the transfections were performed with 0.5 µg of FLAG-Gbeta1, 1.0 µg of PhLP variants, and 1.5 µg of HA-G{gamma} as indicated in Fig. 6A. After 48 h, cells were lysed and extracts were immunoprecipitated with 2.5 µg of anti-CCT{epsilon} antibody (Serotec), the immunoprecipitates were resolved on SDS-PAGE gels and immunoblotted for FLAG-Gbeta1, PhLP-myc, Pdc-myc, or HA-G{gamma}2 using the indicated antibodies as described above. Gbeta1 bands were quantified as described above, and intensities were calculated as a percentage of the control as indicated.

For binding experiments involving endogenous Gbeta, HEK-293 cells were grown in 100-mm dishes and transfected with 6.0 µg of PhLP variant cDNAs as indicated in Fig. 5B. Cells were lysed in 1.2 ml of buffer, and 1 ml was immunoprecipitated with 10 µg of anti-CCT{epsilon} antibody and 60 µl of protein A/G beads. Endogenous Gbeta1 was detected with the anti-Gbeta1 antibody. For binding experiments involving endogenous PhLP, HEK-293 cells were grown in 6-well plates and transfected with 1.0 µg of FLAG-Gbeta1 cDNA as indicated in Fig. 5C. Extracts from two wells were pooled, and 200 µl was immunoprecipitated with 3 µg of anti-CCT{epsilon} antibody. Other immunoprecipitation and immunoblotting procedures were as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of CK2 Phosphorylation of PhLP on CCT and Gbeta{gamma} Binding—To begin to assess the impact of CK2 phosphorylation on PhLP function, the effects of phosphorylation on the binding of PhLP to its two known binding partners, Gbeta{gamma} and CCT, were determined in vitro. Purified recombinant human PhLP was readily phosphorylated by CK2, resulting in a marked reduction in the mobility of the PhLP protein band in SDS-PAGE gels (Fig. 1A). The entire PhLP band was shifted, indicating that phosphorylation was 100% complete under the conditions used. The effects of CK2 phosphorylation on CCT binding was assessed by measuring the ability of PhLP to co-immunoprecipitate CCT from rabbit reticulocyte lysate (18). Human PhLP bound CCT with a high affinity, as evidenced by the fact that addition of only 5 nM PhLP was sufficient to co-immunoprecipitate readily detectible amounts of CCT from the reticulocyte lysate. Under these conditions, CK2 phosphorylation increased the co-immunoprecipitation of CCT by 7-fold (Fig. 1B). In contrast, CK2 phosphorylation had no effect on the ability of PhLP to co-immunoprecipitate purified Gbeta1{gamma}1 (Fig. 1B) or to inhibit association of Gbeta1{gamma}1 with Gt{alpha} and light-activated rhodopsin (Fig. 1C), indicating that phosphorylation did not change the binding of PhLP to Gbeta1{gamma}1.


Figure 2
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  		FIGURE 2.
Mass spectrometric analysis of the CK2 phosphorylation sites of PhLP. A, PhLP was phosphorylated by CK2 in vitro and digested with trypsin, and the resulting peptide fragments were analyzed by LC/MS/MS. CID spectrum of the +2 parent ion corresponding to the m/z of the C-terminal sequence of PhLP plus one phosphate are indicated above the spectrum. The PhLP sequence ends at Asp-301, and the additional residues (smaller font) are part of the linker for the C-terminal myc tag of the recombinant human PhLP. The m/z values corresponding to the b and y ions resulting from fragmentation of this peptide are indicated. The 1764 m/z peak corresponds to the y16 ion with one dehydroalanine generated from the loss of H3PO4 from a phosphoserine during fragmentation. B, CID spectrum of the +2 parent ion corresponding to the m/z of residues 13-32 of PhLP plus one phosphate. The m/z values corresponding to the b and y ions resulting from fragmentation of this peptide are indicated. C, CID spectrum of the +2 parent ion corresponding to the m/z of residues 13-32 of PhLP plus two phosphates. The m/z values corresponding to the b and y ions resulting from fragmentation of this peptide are indicated.

 
Mass Spectrometric Analysis of the CK2 Phosphorylation Sites of PhLP—The phosphorylation sites that could potentially be responsible for the increase in PhLP binding to CCT upon CK2 phosphorylation were identified by mass spectrometry. PhLP was phosphorylated by CK2 in vitro and digested with trypsin, and the resulting peptide fragments were analyzed by electrospray tandem mass spectrometry. Fig. 2A shows the collision-induced dissociation (CID) spectrum of a doubly charged parent ion with an m/z ratio of 1198.5, corresponding to the mass of a tryptic peptide containing the C-terminal residues 287-301 of PhLP plus one phosphate. This CID spectrum showed robust peaks for both the b and y ions corresponding to the sequence of the 287-301 peptide. Of these, the y16 ion had an m/z equal to the loss of a phosphate, and the formation of a dehydroalanine at one of the two serines in this fragment, indicating that either Ser-293 or Ser-296 was phosphorylated in the parent ion. The b2, b4, b6, and b9 ions all had m/z ratios corresponding to the mass of their unphosphorylated fragments, suggesting that Ser-288 and Ser-293 were not phosphorylated. Therefore, the phosphate most likely resided on Ser-296. Accordingly, Ser-296 is within a strong consensus site for CK2 phosphorylation with negatively charged residues at the +1 and +3 positions (28).

One other tryptic fragment with m/z values corresponding to a phosphorylated species was detected and analyzed by CID. This peptide consisted of PhLP residues 13-32 plus one and two phosphates. The spectrum for the singly phosphorylated species yielded few b and y ions, none of which were phosphorylated. However, there were sufficient fragments to confirm the identity of the peptide (Fig. 2B). The same result was obtained with the doubly phosphorylated species. The CID spectrum confirmed the identity of the peptide but did not show any phosphorylated fragments (Fig. 2C). Hence, the four serines of this peptide, serines 18-20 and 25, could all be considered as potential CK2 phosphorylation sites. Of the four, Ser-20 and Ser-25 are within CK2 consensus sites, and phosphorylation of Ser-20 would create a strong consensus site for CK2 phosphorylation of Ser-19 in a doubly phosphorylated species. Similarly, phosphorylation of Ser-19 would make Ser-18 a good CK2 site, although no triply phosphorylated species of the 18-32 peptide were detected. Together, the mass spectrometric data suggest five potential CK2 phosphorylation sites on PhLP: Ser-18, Ser-19, Ser-20, Ser-25, and Ser-296.

Contribution of Specific CK2 Phosphorylation Sites to the PhLP-CCT Interaction—To identify which of these sites is responsible for the phosphorylation-dependent increase in PhLP binding to CCT, each of the five serines identified above was substituted with alanine in various combinations. The resulting PhLP variants were CK2-phosphorylated, and their binding to CCT was determined as described in Fig. 1. Substitution of one residue within the serine 18-20 sequence caused only minor reductions in the phosphorylation-induced increase in binding, whereas substitution of two residues within this sequence resulted in reductions in the phosphorylation-induced binding from 7-fold to ~4-fold (Fig. 3). Replacement of all three serines within this phosphorylation site caused a further reduction in the phosphorylation-induced increase to 3-fold, indicating that multiple phosphorylation events within the serine 18-20 site were responsible for much of the observed increase in PhLP binding to CCT upon CK2 phosphorylation.

With regard to the Ser-25 and Ser-296 sites, replacement of both residues caused a similar modest decrease in binding as was seen with dual substitution within the serine 18-20 site, while replacement of either Ser-25 or Ser-296 along with all three of the Ser-18, Ser-19, and Ser-20 residues was required to completely block the phosphorylation-induced increase in binding. These results show that each of the five serines identified by mass spectrometry can contribute to the phosphorylation-induced increase in PhLP binding to CCT and that no other CK2 phosphorylation sites are involved in this process, suggesting that all the major CK2 phosphorylation sites were identified in the mass spectrometric analysis.

It is important to note that the serine to alanine replacements did not change the binding of unphosphorylated PhLP to CCT significantly (Fig. 3A), indicating that the alanine substitutions did not affect the folding of the PhLP variants nor did they modify the PhLP-CCT interaction significantly. Thus, the loss of the phosphorylation-induced increase in binding with the multiple alanine substitutions could be attributed to an inability of the variants to be phosphorylated by CK2.

Identification of Specific CK2 Phosphorylation Sites in Cells—To assess whether the CK2 phosphorylation sites of PhLP identified in vitro were also phosphorylated in vivo, the decrease in electrophoretic mobility upon CK2 phosphorylation was exploited to detect PhLP phosphorylation events in living cells. In this experiment, PhLP serine to alanine substitution variants with a C-terminal myc tag were transfected into CHO cells. Cells were extracted, and the PhLP was immunoprecipitated and immunoblotted with an antibody to the tag to distinguish the variants from the endogenous PhLP. Substitution of a serine that normally would be phosphorylated by CK2 in wild-type PhLP would be expected to result in an increase in mobility of that PhLP variant. Wild-type PhLP showed decreased mobility of the entire band when compared with an unphosphorylated PhLP standard, indicating that all of the transfected PhLP was phosphorylated in the CHO cells (Fig. 3C, upper panel). The PhLP S20A variant showed two bands, a higher band with the same mobility as wild-type PhLP and a lower band with increased mobility. The ratio of the intensities of the two bands was ~2 to 1, with the higher band having the greater intensity. The PhLP S18A and S19A showed a very small amount of the lower band, whereas S25A and S296A showed no lower bands. The presence of both bands in the S20A variant suggests that phosphorylation of Ser-18 or Ser-19 may be partially impaired when position 20 cannot be phosphorylated. These results indicate that Ser-20 is phosphorylated in cells and that other phosphorylation events might also occur within the serine 18-20 sequence.


Figure 3
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  		FIGURE 3.
Contribution of specific CK2 phosphorylation sites to the PhLP·CCT interaction. A, phosphorylation-induced increase in PhLP binding to CCT is shown for several PhLP variants with the serine-to-alanine substitutions indicated. Binding of CCT to PhLP was determined for each variant as in Fig. 1. Representative immunoblots for the phosphorylated (P) and unphosphorylated (NP) variants are shown. B, the -fold increase in CCT bound upon CK2 phosphorylation of PhLP was calculated by dividing the CCT{epsilon} band intensity of the phosphorylated sample by that of the unphosphorylated sample. The graph gives the average increase ± S.E. from three to five separate experiments. C, the shift in electrophoretic mobility of PhLP upon CK2 phosphorylation was used to determine which of the putative CK2 sites were phosphorylated in cells. CHO cells were transfected with the indicated PhLP variants with C-terminal myc epitope tags. After 48 h, the cells were harvested and extracts were immunoprecipitated and immunoblotted with an antibody to the myc tag. Phosphorylation of the variants was determined by the shift in mobility of the PhLP band compared with wild-type PhLP-myc or purified, unphosphorylated PhLP-myc. D, the electrophoretic mobility of wild-type PhLP and the indicated PhLP variants after CK2 phosphorylation in vitro was also determined to compare the mobility shifts in vitro with those observed in cells. PhLP variants were analyzed by SDS-PAGE gels as in Fig. 1.

 
A similar analysis was done with double and triple serine to alanine substitutions (Fig. 3C, middle panel). The S18A/S19A/S20A variant showed a single lower band compared with wild-type PhLP but that was still higher than the unphosphorylated control. The S18A/S20A and S19A/S20A variants also showed a major lower band, with almost no higher band corresponding to wild-type PhLP. The S18A/S19A variant showed both the higher and lower bands, confirming phosphorylation at Ser-20 and indicating that it is sufficient for the mobility shift. The two bands also suggest that Ser-20 phosphorylation may be impaired in the absence of serine or phospho-serine at position 18 or 19. In the case of the S25A/S296A variant, there was a single higher band with the same mobility as wild-type PhLP, demonstrating that the large decrease in mobility is not a result of Ser-25 or Ser-296 phosphorylation, but rather it stems from at least one phosphorylation event in the Ser-18, Ser-19, and Ser-20 sequence.

The mobility of PhLP variants substituted at four and all five sites was also determined. The S18A/S19A/S20A/S25A variant causes an additional increase in the mobility of the PhLP band to the same mobility as unphosphorylated PhLP, whereas the S18A/S19A/S20A/S296A variant did not increase the mobility beyond that of serines 18-20. The variant in which all five sites were substituted has the same mobility as the S18A/S19A/S20A/S25A variant and unphosphorylated PhLP. These data show that Ser-25 is also phosphorylated in cells, at least in the absence of phosphorylation at serines 18-20, and that Ser-25 phosphorylation causes a small decrease in PhLP mobility. The lack of change in mobility with substitution of Ser-296 did not permit a conclusion to be made about the phosphorylation of this site in cells. Either phosphorylation at Ser-296 did not occur or it did not change the mobility of PhLP in SDS gels.

A very similar pattern of electrophoretic mobility shifts was observed with in vitro CK2 phosphorylation of the PhLP S/A variants (Fig. 3D). S18A/S19A/S20A showed increased mobility compared with wild-type PhLP, and the S18A/S19A/S20A/S25A/S296A variant had the same mobility as unphosphorylated PhLP. The similarities in mobility of the PhLP variants between the in vitro phosphorylation and that found in cells argue that CK2 is responsible for PhLP phosphorylation in vivo, in agreement with previous data indicating that CK2 was the physiologically relevant kinase (14). Importantly, in the absence of CK2 phosphorylation, the S/A variants all had the same mobility as unphosphorylated wild-type PhLP (data not shown), indicating that the differences in mobility were not caused by the alanine substitutions. Together, these data make a strong case for CK2 phosphorylation events within the serines 18-20 and 25 sites in vivo.

Effects of Specific CK2 Phosphorylation Sites on Gbeta{gamma} Expression and Dimer Assembly—It has recently been reported that substitution of all three serine residues in the serine 18-20 sequence blocked the ability of PhLP to enhance the cellular expression of Gbeta{gamma} (24, 25). To further investigate this phenomenon, the CK2 phosphorylation site variants of PhLP were coexpressed with Gbeta1 and G{gamma}2 in HEK-293 cells, and the effects on Gbeta{gamma} expression were measured by immunoprecipitating the G{gamma}2 from cell extracts and immunoblotting for both Gbeta1 and G{gamma}2. Co-expression of the single phosphorylation site variants did not change Gbeta{gamma} expression significantly compared with wild type, nor did co-expression of the S18A/S19A or the S25A/S296A double variants (Fig. 4, A and B). However, co-expression of the S18A/S20A or the S19A/S20A double variants inhibited Gbeta expression by 60-70% and G{gamma} expression by ~50% compared with wild type (Fig. 4A). Likewise, the S18A/S19A/S20A triple variant inhibited Gbeta and G{gamma} expression by 70-80% and 60-70%, respectively (Fig. 4, A and B). Further substitution of Ser-25 and Ser-296 caused no further decreases in Gbeta{gamma} expression (Fig. 4B). These data clearly show that phosphorylation of at least one serine within the serine 18-20 sequence is important for PhLP to assist in the expression of Gbeta{gamma}, with Ser-20 phosphorylation contributing the most to this process. They also show that phosphorylation of Ser-25 and Ser-296 plays no additional role in Gbeta{gamma} expression. Moreover, the significant reduction in Gbeta{gamma} expression by several of the PhLP serine 18-20 variants to levels below those observed with the empty vector indicate that these variants block the ability of endogenous PhLP to support Gbeta{gamma} expression and are thus acting as dominant negative inhibitors of Gbeta{gamma}.

The reason for enhanced Gbeta{gamma} expression in the presence of CK2-phosphorylated PhLP is that phosphorylated PhLP increases the rate of Gbeta{gamma} dimer assembly (24). To determine which phosphorylation sites are critical for PhLP-mediated Gbeta{gamma} assembly, the ability of the PhLP CK2 phosphorylation site variants to catalyze Gbeta{gamma} dimer assembly was determined. All of the double and triple variants of the serine 18-20 sequence were compromised in their ability to assist in Gbeta{gamma} dimer formation compared with wild-type PhLP (Fig. 4C). The S18A/S19A variant was the least compromised, as reflected by an assembly half-life of 99 min compared with 42 min for wild-type PhLP, whereas the S18A/S19A/S20A variant was the most compromised, with a half-life of 284 min (Fig. 4C). The S19A/S20A and S18A/S20A variants showed intermediate half-lives of 153 and 128 min, respectively. In contrast, the S25A/S296A variant was as effective as wild-type PhLP in promoting Gbeta{gamma} assembly with a half-life of 46 min. These Gbeta{gamma} assembly results are qualitatively similar to the Gbeta{gamma} expression data. However, there is one significant quantitative difference in the S18A/S19A variant between the Gbeta{gamma} expression and the Gbeta{gamma} assembly data. Gbeta{gamma} expression was only slightly reduced by the S18A/S19A variant, whereas the rate of Gbeta{gamma} assembly was reduced by >2-fold. This difference can be explained by the 48-h time period over which Gbeta{gamma} expression was measured. It appears that the 2-fold reduction in the rate of Gbeta{gamma} assembly is sufficient to maintain the steady-state Gbeta{gamma} levels achieved in the 48-h expression period to near those found in the presence of wild-type PhLP, whereas the larger reduction in assembly observed with the other Ser-18, Ser-19, and Ser-20 variants is not. Together, the Gbeta{gamma} assembly and expression data indicate that two phosphorylation events in the serine 18-20 sequence are required for PhLP to be fully active in catalyzing Gbeta{gamma} assembly. Phosphorylation at one of the three sites results in partial activity, with Ser-20 phosphorylation conferring the most activity. The results also show that phosphorylation of Ser-25 or Ser-296 has no bearing on Gbeta{gamma} assembly.


Figure 4
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  		FIGURE 4.
Effects of PhLP phosphorylation on Gbeta{gamma} expression and assembly. A, cellular expression of Gbeta{gamma} dimers was determined in the presence of PhLP S18A/S19A/S20A variants. HEK-293 cells were transfected with Gbeta1, HA-tagged G{gamma}2, and the indicated variants. The cells were harvested, and extracts were immunoprecipitated with an antibody to the HA tag. The amount of HA-G{gamma}2 and co-immunoprecipitated Gbeta1 was determined by immunoblotting with anti-HA and anti-Gbeta1 antibodies. A representative blot is shown. The graph gives the average Gbeta1 and HA-G{gamma}2 amounts ± S.E. relative to wild-type PhLP from three separate experiments. Cells in the empty sample were transfected with pcDNA3.1 vector with no PhLP cDNA. B, similar experiments were performed with PhLP variants S25A and S296A separately and in combination with S18A/S19A/S20A. The data are also combined from three separate experiments. C, the rate of nascent Gbeta1{gamma}2 dimer formation in the presence of CK2 phosphorylation site variants of PhLP was determined using a radiolabel pulse-chase assay. Time measurements indicate the sum of the 10-min pulse and the variable chase periods. A representative gel is shown. Band intensities were quantified, and molar ratios of Gbeta1 to HA-G{gamma}2 were calculated and plotted. Lines represent a fit of the data from three separate experiments to a first-order rate equation. Values for t1/2 are shown next to the graph.

 


Figure 5
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  		FIGURE 5.
Gbeta binds CCT in a ternary complex with PhLP. A, binding of Gbeta to CCT was detected by co-immunoprecipitation. HEK-293 cells were transfected with FLAG-Gbeta1, PhLP, Pdc, or HA-G{gamma}2, and cell extracts were immunoprecipitated with an antibody to CCT{epsilon} to bring down CCT complexes. The immunoprecipitates were immunoblotted for Gbeta1, PhLP, Pdc, or G{gamma}2. B, the effects of PhLP on the binding of endogenously expressed Gbeta to CCT were measured by co-immunoprecipitation. HEK-293 cells were transfected with wild-type PhLP, the PhLP S18A/S19A/S20A or {Delta}1-75 variants, or empty vector. Cell extracts were immunoprecipitated with the anti-CCT{epsilon} antibody and immunoblotted for endogenous Gbeta1. A representative immunoblot is shown. Bars in the graph represent the average ± S.E. of the Gbeta band intensity relative to the empty vector control from four separate experiments. C, the effects of Gbeta on the binding of endogenously expressed PhLP to CCT were also measured by co-immunoprecipitation. HEK-293 cells were transfected with Gbeta1, CCT was immunoprecipitated as in panel B, and samples were immunoblotted for endogenous PhLP. A representative immunoblot is shown. Bars in the graph represent the average ± S.E. of the PhLP band intensity relative to the empty vector control from three separate experiments.

 
Gbeta Binds CCT in a Ternary Complex with PhLP—The correlation between the increase in binding of PhLP to CCT upon phosphorylation of serines 18-20 (Fig. 3B) and the necessity of phosphorylation of serines 18-20 for full activity in Gbeta{gamma} assembly (Fig. 4C) suggests that the effects of PhLP phosphorylation on assembly may occur through CCT. However, a role for CCT in Gbeta{gamma} assembly has not been established (24). If CCT does participate in the assembly process, then it must interact with Gbeta, G{gamma}, or both. An interaction between Gbeta and CCT has been observed in yeast protein interaction screens, but no such interaction has been reported in mammalian cells. Therefore, the binding of Gbeta and G{gamma} to CCT was assessed by co-immunoprecipitation of overexpressed Gbeta or G{gamma} in HEK-293 cells. Gbeta co-immunoprecipitated with CCT robustly, to a similar extent as overexpressed PhLP, whereas overexpressed Pdc, which does not bind CCT, was not found in the CCT immunoprecipitate (Fig. 5A). Thus, Gbeta appears to be specifically interacting with CCT under overexpression conditions. In contrast, overexpressed G{gamma} did not co-immunoprecipitate with CCT (Fig. 5A). To determine whether the interaction also occurred with endogenous amounts of Gbeta, the experiment was also done without overexpressing Gbeta. Co-immunoprecipitation of Gbeta with CCT was also observed with endogenous Gbeta, confirming the results of the overexpression experiments (Fig. 5B).

The manner in which PhLP binds CCT at the top of the apical domains without entering the folding cavity (18) suggests that PhLP, Gbeta, and CCT might form a ternary complex in the process of Gbeta{gamma} folding. If such a ternary complex does exist, then PhLP would be predicted to increase the binding of Gbeta to CCT and vice versa. To test this possibility, the effects of PhLP or Gbeta overexpression on the binding of the other to CCT was measured. As predicted, Gbeta overexpression increased the binding of endogenous PhLP to CCT (Fig. 5C). However, PhLP overexpression unexpectedly caused a small but reproducible decrease in Gbeta binding to CCT (Fig. 5B). It is possible that this decrease in Gbeta binding to CCT might be caused by PhLP-catalyzed Gbeta{gamma} assembly and release of the Gbeta{gamma} dimer from CCT. To test this possibility, the effects of two PhLP variants that do not support Gbeta{gamma} assembly on Gbeta binding to CCT were also tested. One variant was PhLP S18A/S19A/S20A, and the other was a truncation variant in which residues 1-75 had been removed (PhLP {Delta}1-75) (24). Both of these variants bind CCT, but they block Gbeta{gamma} assembly in a dominant negative manner (24). Overexpression of either of these variants increased endogenous Gbeta binding to CCT dramatically (Fig. 5B). Thus, it appears that in the absence of serine 18-20 phosphorylation, PhLP forms a ternary complex with Gbeta and CCT that cannot progress in the assembly process. It is interesting to note that the PhLP{Delta}1-75 variant binds Gbeta{gamma} very poorly (24), yet it is still able to stabilize the complex between Gbeta and CCT. This observation indicates that PhLP {Delta}1-75 may do so, more through interactions with CCT than through interactions with Gbeta.

PhLP Phosphorylation Is Required for the Release of Gbeta from CCT and Interaction with G{gamma}—To further investigate the apparent correlation between the destabilization of the PhLP·Gbeta·CCT ternary complex by PhLP phosphorylation and the requirement for PhLP phosphorylation in Gbeta{gamma} assembly, the effects of G{gamma} on ternary complex formation with several PhLP variants was measured. Gbeta was overexpressed in HEK-293 cells with G{gamma} and PhLP variants as indicated, and the amount of Gbeta co-immunoprecipitating with CCT was measured (Fig. 6A). Co-expression of G{gamma} caused a decrease in Gbeta binding to CCT that was intensified by the co-expression of wild-type PhLP. In striking contrast, Gbeta binding to CCT was greatly enhanced by co-expression of PhLP {Delta}1-75 and was completely insensitive to co-expression of G{gamma}. Co-expression of PhLP S18A/S19A/S20A also enhanced Gbeta binding to CCT significantly, and G{gamma} had much less of an effect on binding than with wild-type PhLP. Interestingly, the effects of PhLP {Delta}1-75 and S18A/S19A/S20A on Gbeta binding to CCT in the presence of G{gamma} were quantitatively very similar to their effects on Gbeta{gamma} assembly. PhLP {Delta}1-75 completely blocked Gbeta{gamma} assembly (24) and G{gamma}-mediated dissociation of Gbeta from CCT, whereas PhLP S18A/S19A/S20A decreased the rate of Gbeta{gamma} assembly by 15-fold (24) and G{gamma}-induced dissociation of Gbeta from CCT by 9-fold (compare the Gbeta{gamma} PhLP-WT sample to the Gbeta{gamma} PhLP S18A/S19A/S20A sample in Fig. 6A). From these data, it appears that PhLP phosphorylation contributes to Gbeta{gamma} assembly by enhancing the ability of G{gamma} to release Gbeta from the ternary complex.


Figure 6
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  		FIGURE 6.
PhLP phosphorylation is required for the release of Gbeta from CCT and interaction with G{gamma}. A, the effects of PhLP phosphorylation and G{gamma} co-expression on Gbeta binding to CCT were measured by co-immunoprecipitation. HEK-293 cells were transfected with FLAG-Gbeta1, HA-G{gamma}2, and the PhLP variants as indicated. Cell extracts were immunoprecipitated with an antibody to CCT{epsilon} and then immunoblotted for FLAG-Gbeta. A representative immunoblot is shown. Bars in the graph represent the average ± S.E. of the Gbeta band intensity relative to the Gbeta/PhLP-WT sample from three separate experiments. B, the effects of PhLP phosphorylation on the rate of Gbeta release from CCT were measured using a radiolabel pulse-chase assay. HEK-293 cells were transfected with FLAG-Gbeta1 and the indicated PhLP variants. The pulse-chase assay was performed as in Fig. 4C. After the chase times indicated, cell extracts were immunoprecipitated with antibodies to CCT{epsilon} or Gbeta1. Proteins were resolved by SDS-PAGE, and radiolabeled bands were detected using a PhosphorImager. The Gbeta band intensities were quantified, and ratios of nascent Gbeta1 in the CCT immunoprecipitate versus the total nascent Gbeta in the Gbeta immunoprecipitate were calculated and plotted as a percentage of the ratio at the first time point. Lines represent a fit of the data from 3 separate experiments to a first-order rate equation. Values for t1/2 are shown below the graph. C, the effects of G{gamma} on PhLP-mediated release of nascent Gbeta1 from CCT were measured as in panel B in HEK-293 cells co-expressing HA-G{gamma}2 in addition to FLAG-Gbeta1 and PhLP. D, the ability of G{gamma} to bind CCT was assessed by co-immunoprecipitation. HEK-293 cells were transfected with FLAG-Gbeta1, HA-G{gamma}2, or PhLP variants as indicated. Cells extracts were immunoprecipitated with an antibody to CCT{epsilon} and then immunoblotted for G{gamma}.A representative blot is shown. The Std lane in the CCT IP panel was the lysate from the G{gamma}-transfected cells.

 
There are two possible mechanisms by which phosphorylated PhLP could contribute to G{gamma}-mediated release of Gbeta from CCT. Both involve a conformational change in the ternary complex upon PhLP phosphorylation. First, PhLP phosphorylation could induce a conformation that allows G{gamma} to access Gbeta in the ternary complex and form the Gbeta{gamma} dimer. The Gbeta{gamma} would then be released from CCT. Second, phosphorylation could induce a conformation that releases PhLP·Gbeta from CCT, thereby freeing the G{gamma} binding site on Gbeta for Gbeta{gamma} association to occur. To distinguish between these two mechanisms, the effects of G{gamma} and PhLP overexpression on the rate of dissociation of Gbeta from CCT were measured. In this experiment, cells co-expressing Gbeta with G{gamma}, PhLP, or PhLP S18A/S19A/S20A were pulsed with [35S]methionine for 10 min to label the nascent polypeptides and then were chased with unlabeled methionine. At the times indicated, the cells were lysed and CCT was immunoprecipitated. The co-immunoprecipitating proteins were separated by SDS-PAGE, and the amount of 35S in the Gbeta band was quantified. In the absence of PhLP or G{gamma} co-expression, the dissociation rate of nascent Gbeta from CCT was very slow, with a t1/2 of ~8 h. PhLP co-expression increased the rate by 4-fold to a t1/2 of ~2 h. In contrast, PhLP S18A/S19A/S20A co-expression did not increase the dissociation rate (Fig. 6B). When G{gamma} was co-expressed with Gbeta, the dissociation rate increased by >2-fold to a t1/2 of ~3 h, whereas, when both G{gamma} and PhLP were co-expressed, the t1/2 increased even further to ~2 h, the same value observed in the absence of G{gamma} overexpression (Fig. 6C). When PhLP S18A/S19A/S20A was co-expressed with G{gamma}, there was essentially no Gbeta dissociation, similar to what was seen in the absence of G{gamma} overexpression (Fig. 6C). These effects of G{gamma}, PhLP, and PhLP S18A/S19A/S20A on the dissociation rates are consistent with their effect on the steady-state binding of Gbeta to CCT (Fig. 6A) and further demonstrate that PhLP phosphorylation is required for the release of Gbeta from the ternary complex. These findings are able to distinguish between the two potential mechanisms mentioned above. For example, the enhanced rate of dissociation of Gbeta from CCT upon PhLP overexpression in the absence of G{gamma} overexpression (Fig. 6B) is consistent with the second mechanism in which a phosphorylated PhLP·Gbeta complex would be released prior to G{gamma} binding to Gbeta. This result would not be expected in the first mechanism in which G{gamma} binding would be required for release of Gbeta from CCT. Similarly, the observed lack of increase in the Gbeta dissociation rate upon co-expression of G{gamma} with PhLP would be predicted by the second mechanism but not by the first. On the other hand, the increased release of Gbeta from CCT upon G{gamma} overexpression in the absence of PhLP overexpression is consistent with the first mechanism, but this result could also be explained by the second mechanism if the endogenous PhLP were acting catalytically to release Gbeta from CCT for association with G{gamma}. In this case, the dissociation process would be drawn forward by the formation of the Gbeta{gamma} dimer and its association with G{alpha} and cell membranes (Fig. 7).

To further assess the role of G{gamma} in the release of Gbeta from CCT, the possible association of G{gamma} with Gbeta and PhLP in CCT complexes was determined. G{gamma} was co-expressed with the indicated combinations of Gbeta and the PhLP variants, the CCT complexes were immunoprecipitated, and the samples were immunoblotted for G{gamma}.G{gamma} was not found in any of the CCT immunoprecipitates (Fig. 6D), despite the fact that Gbeta and PhLP could be readily found under these conditions (see Fig. 5). Thus, it appears that G{gamma} does not interact with CCT in any of its complexes with Gbeta and PhLP.

Together, the data in Fig. 6 indicate that PhLP phosphorylation results in the release of a PhLP·Gbeta complex from CCT that can then associate with G{gamma} to form the Gbeta{gamma} dimer. This conclusion is also supported by the previously reported observation that PhLP forms a stable complex with Gbeta that does not include G{gamma} (24).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A model for Gbeta{gamma} Assembly—Recent studies have shown that PhLP acts as an essential chaperone in the assembly of Gbeta{gamma} dimers by binding the Gbeta subunit and thereby allowing G{gamma} to associate with Gbeta (24, 25). Phosphorylation of PhLP at serines 18-20 by CK2 was required for Gbeta{gamma} assembly to occur, yet the means by which phosphorylation of serines 18-20 contributes to assembly was unknown. Moreover, CCT had been implicated in the assembly process, but the results were conflicting (18, 24, 25). The current study provides evidence for a molecular mechanism describing both the role of CCT and PhLP phosphorylation in Gbeta{gamma} assembly (Fig. 7). There are five important steps in this mechanism: 1) the nascent Gbeta polypeptide binds CCT. This is a stable complex that releases Gbeta very slowly in the absence of PhLP. 2) PhLP binds forming a ternary complex. If PhLP is not phosphorylated, then the ternary complex forms in a stable conformation that does not release PhLP·Gbeta, and the Gbeta{gamma} assembly process is blocked. However, if PhLP is dually phosphorylated within the serine 18-20 sequence, then the ternary complex assembles in a conformation that readily releases the PhLP·Gbeta dimer. 3) PhLP·Gbeta dissociates from CCT. The structure of the Pdc·Gtbeta{gamma} complex shows that Pdc binds Gbeta on the opposite face as G{gamma} (29), predicting that the G{gamma} binding site on Gbeta would be free in the PhLP·Gbeta dimer. 4) G{gamma} binds Gbeta forming a PhLP·Gbeta{gamma} complex. This complex is stable with a 100 nM binding affinity (11). However, both the G{alpha} binding site and the membrane association surface of Gbeta{gamma} overlap extensively with the PhLP binding site (11); therefore in the presence of G{alpha} and membranes, PhLP would be expected to be released from Gbeta{gamma}.5)Gbeta{gamma} associates with G{alpha} and/or the endoplasmic reticulum membrane and is transported to the plasma membrane (30). PhLP is then free to catalyze another round of Gbeta{gamma} assembly.


Figure 7
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  		FIGURE 7.
CK2 phosphorylation-dependent release model of Gbeta{gamma} assembly. A model is proposed in which nascent Gbeta forms a ternary complex with CCT and PhLP. If PhLP is not phosphorylated, the ternary complex is stable and PhLP·Gbeta is not released from CCT. If PhLP is phosphorylated, the ternary complex is destabilized, possibly by electrostatic repulsion between the phosphates in the serine 18-20 phosphorylation site and negatively charged residuesonthe CCT{alpha} or -{epsilon} apical domains. Once released, the PhLP·Gbeta complex binds G{gamma}, forming the Gbeta{gamma} dimer. The dimer then associates with G{alpha} and membranes in a manner yet to be defined. In the process, PhLP is released to catalyze another round of dimer formation. The approximate position of the serine 18-20 phosphorylation site is depicted by a red oval marked `P.' The relative amount of positive and negative charges on the CCT apical domains that contact the PhLP N-terminal domain is also indicated. See text for details.

 
This model readily explains the dominant negative effect of the PhLP S18A/S19A/S20A and PhLP {Delta}1-75 variants. These variants form PhLP·Gbeta·CCT ternary complexes that do not release PhLP·Gbeta for G{gamma} binding. Such stable ternary complexes would also block the endogenous, phosphorylated PhLP from forming competent ternary complexes capable of releasing PhLP·Gbeta for G{gamma} binding. Previous explanations of the dominant negative effect of PhLP S18A/S19A/S20A, which postulated that unphosphorylated PhLP would block Gbeta and G{gamma} association with CCT (25) or that unphosphorylated PhLP would form a PhLP·Gbeta complex that would not accept G{gamma} (24), are incomplete.

Phosphorylation-induced Conformational Changes—One apparent inconsistency in the data is that CK2 phosphorylation of PhLP increased its binding to CCT in the absence of Gbeta (Fig. 1), yet PhLP phosphorylation was necessary for the release of PhLP·Gbeta from CCT in the presence of Gbeta (Figs. 4, 5, 6). The difference between these observations may stem from differences in the structures of the PhLP·CCT and PhLP·Gbeta·CCT complexes. Clues regarding the nature of the phosphorylation-dependent changes in these structures may be gleaned from the cryoelectron microscopic studies of the unphosphorylated PhLP·CCT complex (18). In this complex, PhLP was shown to interact in two distinct conformations at the top of the CCT toroid, contacting only the CCT apical domains (18). In one conformation, the N-terminal phosphorylation site of PhLP was in close proximity to the CCT{alpha} and -{epsilon} apical domains and in the other conformation the phosphorylation site was in close proximity to the CCT{zeta} and -beta apical domains. The binding surfaces of all eight apical domains are dominated by charged and polar residues (31) with the CCT{alpha} and -{epsilon} binding surfaces having a high distribution of negative charge, whereas the CCT{zeta} binding surface exhibits an extensive positively charged patch. The serine 18-20 phosphorylation site of PhLP is harbored within a sequence (18SSSDEDESD26) that is already very negatively charged. The addition of phosphates within this sequence would create an extremely high concentration of negative charge that would interact effectively with the positively charged patch of CCT{zeta}. In the absence of Gbeta, phosphorylation could favor the conformation that brings the PhLP phosphorylation site in close proximity to the CCT{zeta} apical domain, increasing the binding of PhLP to CCT. In the presence of Gbeta, it is possible that interactions with Gbeta may limit the ability of PhLP to rotate on the top of the CCT toroid. Thus, the phosphorylation site may be fixed in close proximity to the CCT{alpha} and -{epsilon} apical domains, causing electrostatic repulsion between the negative charges on the CCT{alpha} and -{epsilon} binding surfaces and the PhLP phosphorylation site. This repulsion might destabilize the ternary complex and allow the release of the PhLP·Gbeta complex. Further studies will be required to test the validity of this structural model.

Regulation of CK2 Phosphorylation of PhLP—Given the essential role of CK2 phosphorylation of PhLP in Gbeta{gamma} dimer formation, an important issue yet to be addressed is the regulation of this phosphorylation event. CK2 is a constitutively active kinase with many protein substrates (32). Determination of which substrates are phosphorylated and when phosphorylation occurs appears to be controlled by regulated expression and assembly of the CK2 {alpha}2beta2 tetramer and by the association of different CK2 binding partners (32). In the case of PhLP, CK2 phosphorylation occurs within the first 30 min of its synthesis (data not shown), and it remains completely phosphorylated under the cell culture conditions used here (Fig. 2). It is not clear from the current data whether phosphorylation occurs prior to or after association of PhLP with CCT (Fig. 7). In mouse tissues, PhLP was also completely phosphorylated in brain and heart but was mostly unphosphorylated in the adrenal gland (14). It is possible that Gbeta{gamma} assembly is a continuous process in some cell types, whereas in others assembly is highly regulated, only occurring under certain conditions that promote CK2 phosphorylation of PhLP.

These investigations into the mechanism of PhLP-mediated Gbeta{gamma} assembly and its regulation by CK2 phosphorylation suggest that PhLP and its interactions with Gbeta and CCT could be targeted by therapeutics to control the levels of Gbeta{gamma} expression and thus the degree of G protein signaling within the cell, perhaps providing additional tools to treat the myriad of G protein-linked diseases.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant EY12287 and National Science Foundation Grant MCB-0131361 (to B. M. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, C-100 BNSN, Brigham Young University, Provo, UT 84602. Tel.: 801-422-2785; Fax: 801-422-0153; E-mail: barry_willardson{at}byu.edu.

2 The abbreviations used are: G{alpha}, {alpha} subunit of a heterotrimeric G protein; G, heterotrimeric GTP-binding protein; Pdc, phosducin; PhLP, phosducin-like protein; CCT, cytosolic chaperonin containing tailless complex polypeptide 1; CK2, protein kinase CK2; LC/MS/MS, liquid chromatography coupled with electrospray tandem mass spectrometry; CID, collision-induced dissociation; CHO, Chinese hamster ovary cell; HEK-293 cells, human embryonic kidney 293 cells; HA, hemagglutinin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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