Alternative splicing of the porcine glycogen synthase kinase 3β (GSK-3β) gene with differential expression patterns and regulatory functions

Abstract

Background: Glycogen synthase kinase 3 (GSK3α and GSK3β) are serine/threonine kinases involved in numerous cellular processes and diverse diseases including mood disorders, Alzheimer's disease, diabetes, and cancer. However, in pigs, the information on GSK3 is very limited. Identification and characterization of pig GSK3 are not only important for pig genetic improvement, but also contribute to the understanding and development of porcine models for human disease prevention and treatment.

Methodology: Five different isoforms of GSK3β were identified in porcine different tissues, in which three isoforms are novel. These isoforms had differential expression patterns in the fetal and adult of the porcine different tissues. The mRNA expression level of GSK3β isoforms was differentially regulated during the course of the insulin treatment, suggesting that different GSK3β isoforms may have different roles in insulin signaling pathway. Moreover, GSK3β5 had a different role on regulating the glycogen synthase activity, phosphorylation and the expression of porcine GYS1 and GYS2 gene compared to other GSK3β isoforms.

Conclusions: We are the first to report five different isoforms of GSK3β identified from the porcine different tissues. Splice variants of GSK3β exhibit differential activity towards glycogen synthase. These results provide new insight into roles of the GSK3β on regulating glycogen metabolism.

Figure 1. Alignment of amino acid sequences of the isoforms 1, 2, 3, 4, and 5 of GSK3β gene of porcine.

GSK3β is spliced to five different kinds of mRNAs: GSK3β1, GSK3β2, GSK3β3, GSK3β4 and GSK3β5, which encode five proteins with 420, 433, 399, 387 and 400 amino acids, respectively. GSK3β2 have a 13 amino acid insert in the kinase domain. GSK3β3 and GSK3β4 contain the identical serine/threonine kinase domain but vary in their N-terminus. GSK3β5 lacks a 20 amino acid (203–222) in the kinase domain inculding three important phosphorylation site Lys 205, Tyr 216, Tyr 220.

Figure 2. Schematic representation of the genomic and alternative splicing of porcine GSK3β.

Line above shows the genomic contig from genome database. The exons and introns are represented by boxes and straight lines, respectively. GSK3β is spliced to five different kinds of mRNAs: GSK3β1, GSK3β2, GSK3β3, GSK3β4 and GSK3β5, respectively. GSK3β2 contained an additional exon8 b (yellow), GSK3β3 contained an additional exon10 b (blue), and GSK3β4 lacks exon10 (green) and GSK3β5 lacks 60 nucleotides in exon6 (red), resulting in a 20 amino acid extraction in the kinase domain. The sequences of porcine GSK3β1, 2, 3, 4 and 5 were deposited in GenBank (Accession No. JN387128 to JN387132).

Figure 3. Expression analysis of GSK3β isoforms in porcine and mouse different tissues.

(A) Expression analysis of porcine GSK3β isoforms in different fetal and adult tissues in Meishan pigs by RT-PCR and beta-actin was used as a control. PCR without cDNA template (water) served as negative controls. (B) Expression analysis of mouse GSK3β1 and GSK3β2 in different tissues by RT-PCR and beta-actin was used as a control.

Figure 4. The tissue distribution of porcine GSK3β mRNA was assessed by quantitative real-time PCR.

Error bars indicate the SD (n = 3) of relative mRNA expression levels of GSK3β to beta-actin, determined by qRT-PCR. The values were normalized to endogenous beta-actin expression.

Figure 5. GSK3β protein expression in porcine tissues.

Immunohistochemical analysis of porcine GSK3β using human anti-GSK3β in liver (A), spleen (C), testis (E) tissues and pig kidney epithelial cells (B) as compared with controls (D, and F). Arrows indicate positive staining.

Figure 6. Differential time requirement for the change in mRNA expression level of different GSK3β isoforms in response to treatment with insulin.

(A) Total GSK3β mRNA and protein expression was detected by semi-quantitative RT-PCR and western blotting, and Beta-actin was used as a control. (B) PK-15 cells were incubated in 100 nM insulin for 0, 5, 10, 15, 20 or 30 min after which cells total RNA was extracted for determining the expression of different GSK3β isoform by qRT-PCR. The values were normalized to endogenous beta-actin expression and the value of GSK3β2 after insulin incubation in 30 min was arbitrarily set to 1. Significant levels were analyzed by T-test. **, p < 0.01; *, p < 0.05.

Figure 7. Effects of different GSK3β isoforms overexprssion on glycogen synthase activity.

(A) Semi-quantitative RT-PCR analysis and Beta-actin was used as a control. (B, C) Quantitative real-time PCR was performed. The expression level of GYS1 and GYS2 was normalized to endogenous Beta-actin mRNA levels. Significant levels were analyzed by T-test. Data are expressed as means±SEM of three separate experiments. (D) PK-15 cells were tranfected in different GSK3β isoforms for the 48 h after which cells were lysed and we measured glycogen synthase activity in cells by assaying the incorporation of radioactive UDP-glucose into glycogen in the presence or absence of the allosteric activator glucose 6-phosphate (G6P).

Figure 8. Regulation of glycogen synthase phosphorylation and activity following over-expression of different GSK3β isoforms.

(A) Phosphorylation of pGSK3β (Ser9) and dephosphorylation of pGSK3β (Tyr216) levels in PK-15 cells transfected with different GSK3β isoforms. Equal amounts of lysate protein from cells were immunoblotted with antibody against phospho-Ser9-GSK3β, phospho-Tyr216-GSK3β and total GSK3β protein. *: Endogenous GSK3β. The blots are representative from up to three separate experiments. β-actin was used as the loading control. (B) Regulation of glycogen synthase phosphorylation following expression of different GSK3β isoforms induced by insulin (100 nM). PK-15 cells transiently transfected with pcDNA3.1-GSK3β1, 2, 3, 5 or the empty pcDNA3.1 expression vector. 24 h post-infection cells were incubated in the absence or presence of 100 nM insulin for 10 min. Following these incubations, cells were lysed and immunoblotting with antibodies against phosphor-Ser641-GS. β-actin was used as the loading control.

Figure 9. The subcellular localization of porcine GSK3β1 and GSK3β5 proteins in PK15 cells.

The recombinant plasmid pEGFP-GSK3β1 and GSK3β5 were transiently transfected into PK15 cells using the Lipofectamine 2000 reagent. The GSK3β1 and GSK3β5-GFP fusion proteins were all distributed in localize both in cytoplasm and nuclei (excited at 488 nm; B, E), and nuclei were stained with DAPI (excited at 360 nm; A, D). The fluorescent signals were analyzed by confocal microscopy. The overlay images were produced by merging all three signals together (C, F).