Expression of glycogen synthase kinase-3 isoforms in mouse tissues and their transcription in the brain

Abstract

Glycogen synthase kinase-3α and -3β (GSK-3α and -3β) are multi-substrate, serine/threonine-specific kinases that can phosphorylate microtubule-associated protein tau and other neuronal proteins. In this study, the expression level and mRNA distribution of two GSK-3 isoforms, GSK-3α and -3β in mice were investigated. Northern blot analyses indicated that GSK-3α mRNA is encoded by a 2.5-kb transcript in adult tissues, whereas a 4.1-kb transcript was found in neonatal tissues. The GSK-3β mRNA is encoded by a 1.6-kb transcript in the testis and a 7.6-kb transcript in the brain, and in many other adult tissues, but not neonatal tissues. Western blot analyses demonstrated that GSK-3β protein was mainly expressed in the brain and heart, whereas GSK-3α was highly expressed in the brain, heart, and testis. A non-radioactive in situ hybridization study using specific digoxigenin-labeled RNA probes showed that GSK-3α and -3β mRNAs were found in many brain regions, and were especially abundant in the hippocampus, cerebral cortex, and the Purkinje cells of the cerebellum. This implies the importance of GSK-3α and -3β for brain function. The differential expression of GSK-3α and -3β mRNAs as well as proteins in other tissues indicate that they play different roles in cellular functions and the developmental process.

Introduction

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine-specific kinase, which was initially found to be able to phosphorylate and thereby inactivate glycogen synthase (Embi et al., 1980, Hemmings et al., 1981). Two isoforms, designated GSK-3α and GSK-3β, are monomeric enzymes encoded by two distinct genes (Woodgett, 1990). In humans, the two corresponding genes have been mapped to chromosomes 19q12.3 and 3q13.3, respectively (Hansen et al., 1997, Shaw et al., 1998). GSK-3α encodes a 51-kDa protein, while GSK-3β encodes a 47-kDa protein, and overall they are 85% homologous to each other. The catalytic kinase domains of the two proteins show 95% identity, indicating that these two isoforms may have similar spectra of enzymatic activities. The sequences outside of the kinase domain are, however, quite distinct, with a glycine/serine rich segment found only at the N-terminal region of GSK-3α (Woodgett, 1990, Woodgett, 1994).

A previous study has shown that GSK-3 RNA and proteins are expressed in many tissues but are particularly rich in the brain (Woodgett, 1990). Several findings have shown that GSK-3 can phosphorylate nuclear transcriptional factors and cytoplasmic proteins. These factors include NF-AT (Beals et al., 1997), c-jun (Nikolakaki et al., 1993), myc proteins (Saksela et al., 1992, Henriksson et al., 1993, Pulverer et al., 1994), ATP citrate lyase (Ramakrishna and Benjamin, 1985), and translation factor eIF2b (Welsh et al., 1997). The homolog of GSK-3 is essential for control of body pattern development in Drosophila and in Xenopus (Ruel et al., 1993, He et al., 1995). These findings suggest that GSK-3 is a key kinase, critically involved in multiple cellular processes such as embryonic development, cell differentiation, ontogenesis, and apoptosis (Frame and Cohen, 2001).

There is increasing evidence showing that GSK-3 is one of the kinases also crucially involved in the hyper-phosphorylation of tau in Alzheimer's disease (AD). GSK-3, especially the β-isoform, is associated with the paired helical filaments (PHF) found in the AD brain (Shiurba et al., 1996, Yamaguchi et al., 1996, Pei et al., 1997). The major component of PHF is the hyper-phosphorylated tau protein (Ihara et al., 1986). The levels of GSK-3β are also increased in the AD brain (Imahori and Uchida, 1997, Pei et al., 1997, Imahori et al., 1998). GSK-3β has been shown to phosphorylate tau in vitro on several serine and threonine residues known to be phosphorylated in PHF-tau (Lovestone et al., 1994, Mulot et al., 1994, Anderton et al., 1995).

Recent studies have shown the developmental expression and localization of GSK-3β in the rat brain (Takahashi et al., 1994, Leroy and Brion, 1999, Takahashi et al., 2000), but overall, little information is available on the distribution of GSK-3α and -3β mRNAs in the brain. In order to further understand the different roles of these two GSK-3 isoforms, we have carried out an expression analysis of GSK-3α and -3β in mouse tissues and their mRNA localization in the brain.