Mechanism of Assembly of G Protein βγ 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,Barry M Willardson

doi:10.1074/jbc.m601590200

Georgi L Lukov, Christine M Baker + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m601590200

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Abstract

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein βγ subunit complex (Gβγ) that has been recently shown to catalyze the formation of the Gβγ 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 Gβγ 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 Gβγ-assembly process. The results reported here delineate a mechanism for Gβγ assembly in which a stable ternary complex is formed between PhLP, the nascent Gβ subunit, and CCT that does not include Gγ. PhLP phosphorylation permits the release of a PhLP·Gβ intermediate from CCT, allowing Gγ to associate with Gβ in this intermediate complex. Subsequent interaction of Gβγ with membranes releases PhLP for another round of assembly.

Highlights

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 Phosducin-like protein (PhLP) binding to containing tailess complex polypeptide 1 (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
A model for G␤␥ Assembly—Recent studies have shown that PhLP acts as an essential chaperone in the assembly of G␤␥ dimers by binding the G␤ subunit and thereby allowing G␥ to associate with G␤ [24, 25]
The current study provides evidence for a molecular mechanism describing both the role of CCT and PhLP phosphorylation in G␤␥ assembly (Fig. 7)

Summary

EXPERIMENTAL PROCEDURES

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). 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 Ni2ϩ affinity chromatography as described previously [11]. The cells were harvested 48 h later in 200 ␮l of immunoprecipitation buffer [24], and the PhLP-myc was immunoprecipitated from the lysate with 3 ␮g of anti-cmyc antibody and 30 ␮l of Protein A/G beads as described previously [17, 24]. G␤␥ Expression Measurements—HEK-293 cells were plated in 6-well plates so that they would be 70 – 80% confluent the following day They were co-transfected with 1 ␮g of each of the PhLP-myc, HA-G␥2, and G␤1 cDNAs using Lipofectamine Plus reagent.

CCT binding experiments involving

DISCUSSION