Pannexin 1 mutation found in melanoma tumor reduces phosphorylation, glycosylation, and trafficking of the channel-forming protein

Abstract

Pannexin 1 (PANX1) is a glycoprotein that forms large pore channels capable of passing ions and metabolites such as ATP for cellular communication. PANX1 has been implicated in many diseases including breast cancer and melanoma, where inhibition or deletion of PANX1 reduced the tumorigenic and metastatic properties of the cancer cells. We interrogated the effect of single amino acid changes in various PANX1 domains using naturally occurring variants reported in cancer patient tumors. We found that a previously reported variant (Q5H) is present in cancer cells, but was not different from the wild type (Q5) in glycosylation, trafficking, or channel function and did not affect cellular properties. We discovered that the Q5H variant is in fact the highly conserved ancestral allele of PANX1 with 89% of humans carrying at least one Q5H allele. Another mutated form Y150F, found in a melanoma patient tumor, prevented phosphorylation at Y150 as well as complex N-glycosylation while increasing intracellular localization. Sarcoma (SRC) is the predicted kinase to phosphorylate the Y150 residue, and its phosphorylation is not likely to be constitutive, but rather dynamically regulated. The Y150 phosphorylation site is the first one reported to play a role in regulating posttranslational modifications and trafficking of PANX1, with potential consequences on its large-pore channel structure and function in melanoma cells.

FIGURE 1:

Impact of PANX1 mutations on protein banding and cell-surface localization in NRK cells. (A) Schematic of PANX1 polypeptide demonstrating the location of PANX1 variants Q5H, Y150F, G168E, T176I, H190Y, S239L, and Q264*. The schematic was generated using Protter (Omasits et al., 2014). NT, amino-terminus; TM, transmembrane; EL, extracellular loop; CT, carboxyl-terminus; IL, intracellular loop. (B) NRK cells were transfected to transiently overexpress selected PANX1 variants and PANX1 banding pattern was assessed 72 h posttransfection and compared with WT PANX1 (Q5). Immunoblotting with anti-PANX1-CT antibody revealed Y150F disrupts the normal banding pattern of PANX1, resulting in primarily Gly1 with minimal Gly0 and Gly2 species. EV, empty vector. (C) PANX1 protein was not detected in NRK cells transfected with Q264*-encoding vectors using two different antibodies against the C-Terminus or N-Terminus. The molecular weight of Q264* is expected to be approximately around 29 kDa. GAPDH was used as a loading control. Protein sizes are noted in kDa. (D) Immunofluorescence analyses revealed that most variants appeared to localize to the cell surface on ectopic expression in NRK cells, except for Y150F that had an increased intracellular profile. Bar: 10 µm.

FIGURE 2:

Stable overexpression of Q5H in Hs578T(KO) cells does not differ in dye uptake, migration, or cell growth from Q5 controls. (A) Stable clones of Hs578T(KO) overexpressing Q5 or Q5H at similar protein levels were generated. GAPDH used as loading control. One-way ANOVA (letters denote significantly different groups). (B) Immunofluorescence analyses (PANX1, green) demonstrate that Q5 and Q5H both localize at the cell surface in stable clones. Nuclei are in blue. Scale bar: 20 µm. (C) Q5H did not differ significantly from Q5 in basal uptake of YO-PRO1 dye but both were significantly higher than nontransfected controls (KO). One-way ANOVA (letters depict significant differences). (D) Cell growth assay was performed by assessing the number of viable cells that excluded trypan-blue dye. Cell counts did not differ significantly between Hs578T(KO) stably overexpressing Q5 and Q5H and nontransfected controls. Two-way ANOVA (NS, not significant). (E) Cells were grown to confluence and a scratch was made on which the area cells migrated into after 15 h was recorded. Migration did not differ significantly between Hs578T(KO) stably overexpressing Q5 and Q5H. N = 3, paired t test (NS, not significant).

FIGURE 3:

Higher Q5H allele frequency than Q5 in global cohorts. (A) Allele frequency of Q5H found in diverse populations (based on ancestry). Data were extracted from the ExAC Browser Beta. (B) The genotype frequency of individuals sequenced in the 1000 Genomes Project (phase 3) demonstrates that most individuals (∼89%) possess at least one allele copy of Q5H. (C) Genotype frequencies of Q5 and Q5H in different populations from the 1000 Genomes Project (phase 3) shows that the prevalence of each allele varies in different ethnic groups. (D) Ensembl phylogenetic context alignment revealed Q5H is a highly conserved site amongst vertebrate species with humans being the only species with Q5 (according to RefSeq NCBI).

FIGURE 4:

Location of Q5 and Y150 residues in 2D and 3D structures of PANX1. (A) PANX1 2D polypeptide demonstrating the location of PANX1 Q5 and Y150 residues. (B) Monomeric 3D structure indicating the location of Y150 in the intracellular loop adjacent to a region (dashed line) not resolved in the 3D structure and in close proximity of a region (positions 322–329) of the carboxyl-terminus of the same monomer. NT, amino-terminus; TM, transmembrane; EL, extracellular loop; CT, carboxyl-terminus; IL, intracellular loop. The structure of the region corresponding to Q5 in the NT has not been published. (C, D) Solvent-excluded molecular surface and ribbon representations (transparency) of the side and intracellular views of the heptameric PANX1 channel showing the location of Y150 in each subunit within the inner linings of the pore. The hydroxyl-F side chain of Y150 is located inward pointing to the center of the channel. The 2D and 3D figures were generated using Protter (Omasits et al., 2014) and UCSF Chimera/ChimeraX (Pettersen et al., 2004), respectively. The cryo-EM 3D structure of PANX1 (PDB: 6m66) was used as a model for all the visualizations (Jin et al., 2020).

FIGURE 5:

Q5H and Y150F do not affect PANX1 channel currents in Hs578T(KO) cells. (A) Averaged current-voltage relationships recorded in the presence or absence of PANX1 blocker CBX (red) in Hs578T (WT) and Hs578T (KO) cells expressing Q5, Q5H, or Y150F (n = 11 cells for each condition). (B) Summary of ramp currents recorded at +100 mV from WT, nontransfected KO and KO cells expressing Q5, Q5H, and Y150F (n = 11 cells per condition, *p < 0.05; NS, not significant; one-way ANOVA with means compared with KO). (C) Summary of currents recorded at –60 mV from WT, nontransfected KO, and KO cells expressing Q5, Q5H, and Y150F (n = 11 cells for each condition; NS, not significant; one-way ANOVA).

FIGURE 6:

Y150F produces a Gly1 species that is partially resistant to EndoH deglycosylation. (A) Hs578T(KO) cells and A375P(KO) cells devoid of PANX1 were used to transiently overexpress EV, Q5, Q5H, and Y150F and assessed for changes in PANX1 banding pattern via immunoblotting. Western blot analyses revealed that Y150F disrupted the normal banding pattern of PANX1 in both cell lines, resulting in primarily Gly1 species. Gly0 (nonglycosylated), Gly1 (high mannose), Gly2 (complex glycosylation). (B) PNGase F digestion of protein lysates from Hs578T(KO) cells transiently overexpressing PANX1 variants and controls (N = 3). EndoH digestion of protein lysates from Hs578T(KO) transiently overexpressing PANX1 variants and controls resulted in a band shift of Gly1 species of Q5 and Q5H to Gly0 band. In contrast, Y150F was partially resistant to EndoH digestion, even when the amount of Endo H was doubled (++). GAPDH was used as a loading control. (C) Densitometric analysis of Western blots derived from deglycosylation experiments. Integrated intensity values were tested for normality with a Shapiro–Wilk test and converted -log10 for statistical analysis. Comparisons between treatments (+) and controls (–) were performed in each glyco-species individually using multiple t tests with the Holm–Sidak method. No statistical significance was observed (p > 0.05). Representative blots of N = 3.

FIGURE 7:

Y150 is a novel tyrosine phosphorylation site of PANX1. (A) Y150 is predicted to be a tyrosine-phosphorylation site by NetPhos3.1 Server. NetworKIN server predicted kinases of the Y150 site and the top candidate was SRC kinase (0.46 score). (B) PANX1 was IP from Hs578T(KO) transiently overexpressing EV, Q5, Q5H, or Y150F and bands from an SDS–PAGE gel representing Gly0, Gly1, and Gly2 of PANX1 were sent for ESI-MS processing and analysis. (C) Analyses of ESI LC MSMS data demonstrating identification of Y150-containing PANX1 peptides; –10logP Score: ions score, where P is the probability that the observed match is a random event; higher numbers identify the likelihood of a true match.

FIGURE 8:

SRC kinase inhibition with PP2 reduces Gly1 species and decreases cell surface localization of PANX1. (A) NRK cells overexpressing PANX1 WT (Q5), Y150F or Y150A were treated with PP2 to inhibit Src kinases, and the inactive analog PP3 was used as a control. There was no decrease in total protein levels of PANX1 with PP2 treatment between mutants; however, kinase inhibition produced a significant decrease in the Gly1 species compared with control in PANX1 WT expressing cells (p < 0.05), with no effect on the nonphosphorylated mutants (Y150A and Y150F). (B) Protein levels were quantified from representative blots (N = 3) and a two-way ANOVA was performed (*p < 0.05). (C) NRK cells overexpressing PANX1 WT exhibited a more intracellular PANX1 pattern when treated with PP2 compared with the primarily cell surface localization seen in control PP3 cells.

FIGURE 9:

Impact of Y150 PANX1 mutations on protein banding and cell-surface localization in NRK and HS578T-PANX1-KO cells. (A) NRK and HS578T-PANX1 KO cells were transfected with 1 µg of DNA from either WT PANX1 (Q5), Y150 mutants unable to be phosphorylated (Y150F and Y150A) or Y150 phosphomimetics (Y150D and Y150E); 48 h post-transfection, all Y150 mutants disrupted the normal PANX1 banding pattern. Y150F and Y150A showed a loss of Gly2 and mainly Gly1 and Gly0. Y150D and Y150E mutants were toxic to the cells; however, protein that was able to be isolated demonstrated mainly Gly1 banding. (B) Immunoflourescence of all Y150 mutants 48 h post-transfection showed disruption of WT PANX1 localization within both NRK and HS578T-PANX1-KO cells. Y150F and Y150A demonstrate a mainly intracellular pattern of PANX1 compared with the cell surface localization of PANX1 WT. Y150D and Y150E mutants caused cell death, so localization is difficult to interpret. (C) EndoH digestion of protein lysates from NRK and Hs578T(KO) overexpressing WT PANX1 resulted in a shift for some of Gly2 and all the Gly1 species to the Gly0 band. Cells overexpressing Y150F and Y150A showed a partial shift of Gly1 to the Gly0 species, but a portion of Gly1 in these mutants remained partially resistant to EndoH digestion in both cell types (even in the presence of excess enzyme).