Protein Kinase, Amp-Activated, Noncatalytic, Gamma-3

Description

The AMP-activated protein kinase (AMPK) cascade plays an important role in the regulation of energy homeostasis within the cell. AMPK is a heterotrimer composed of 1 catalytic (alpha; see PRKAA1, 602739) and 2 regulatory (beta, see PRKAB1, 602740; and gamma, see PRKAG1, 602742) subunits. All 3 subunits are required to yield significant kinase activity (Cheung et al., 2000).

Cloning and Expression

By screening a human skeletal muscle cDNA library with the C-terminal 201 amino acids of rat beta-1, Cheung et al. (2000) isolated the gamma-3 isoform of the AMP-activated protein kinase. The gamma-3 cDNA encodes a protein of 492 amino acids, with a calculated molecular weight of 55 kD confirmed by SDS-PAGE. The gamma-3 isoform has a long (150-amino acid) N-terminal extension not present in gamma-1; the gamma-2 isoform (602743) has a similar extension. In contrast, the respective C termini of gamma-2 and gamma-3 share significant amino acid sequence identity with gamma-1, with gamma-1 and gamma-3 sharing 63% identity. Northern blot analysis detected strong expression of a 2.4-kb gamma-3 transcript in skeletal muscle, with weak expression in heart and pancreas. A transcript of approximately 4.4 kb was also detected ubiquitously, again with highest expression in skeletal muscle. Cheung et al. (2000) found no evidence for any selective association between the alpha-1 and alpha-2 (600497) isoforms and the various gamma isoforms, but the AMP dependence of the kinase complex was markedly affected by the identity of the gamma isoform present. Complexes containing gamma-2 showed the greatest AMP dependence, while those containing gamma-3 showed the lowest; those containing gamma-1 showed an intermediate effect.

Using human skeletal muscle cDNA, Milan et al. (2000) determined the cDNA sequence of human PRKAG3 by RT-PCR and 5-prime RACE analysis. The protein has 2 regions: pig and human PRKAG3 residues 1 to 159 share 65% identity, whereas residues 160 to 464 share as much as 97% identity. The latter region contains 4 cystathionine beta-synthase domains, a characteristic shared with other AMPK-gamma sequences. Northern blot analysis of human PRKAG3 demonstrated distinct muscle-specific expression, whereas PRKAG1 and PRKAG2 were widely expressed. Milan et al. (2000) noted that the human full-length PRKAG3 cDNA sequence reported by Cheung et al. (2000) encodes an additional 25 amino acids in the N-terminal region and a different sequence in the C-terminal end relative to the sequence described by them. Milan et al. (2000) suggested that alternative splicing occurs in the 5-prime region of the gene, and that experimental data are needed to determine which of the ATG codon(s) are used for initiation of translation. With regard to the difference in the C-terminal end, Milan et al. (2000) believed their sequence to be the correct one as it is consistent with the human genomic sequence as well as the pig coding sequence.

Mapping

The International Radiation Hybrid Mapping Consortium mapped the PRKAG3 gene to chromosome 2 (SHCG-52801).

Molecular Genetics

In 2 probands and 4 additional family members, Costford et al. (2007) identified an R225W mutation in the PRKAG3 gene (604976.0001) that is homologous to the naturally occurring R200Q substitution in Hampshire pigs (see ANIMAL MODEL). In vitro studies showed that the R225W mutation resulted in significantly increased basal and AMP-activated AMPK activity (about 2-fold) in skeletal muscle. Muscle fibers from mutation carriers had approximately 90% more muscle glycogen content than controls (p = 0.002) and approximately 30% decreased levels of intramuscular triglyceride. Levels of lipids, glucose, insulin, and HbA1c were normal. These findings were consistent with findings in Hampshire pigs with the homologous variant. Costford et al. (2007) concluded that the gain-of-function R225W variant results in increased glycogen content in skeletal muscle in humans. The findings also indicated that the PRKAG3 gene plays an important regulatory role in human muscle energy metabolism.

Animal Model

A high proportion of purebred Hampshire pigs carry the dominant RN- mutation, which causes high glycogen content in skeletal muscle. The mutation has beneficial effects on meat content but detrimental effects on processing yield. Milan et al. (2000) used a positional cloning approach to identify an arg200-to-gln (R200Q) substitution in the PRKAG3 gene, which encodes a muscle-specific isoform of the regulatory gamma subunit of AMPK. Loss-of-function mutations in the homologous gene in yeast (SNF4) cause defects in glucose metabolism, including glycogen storage. The muscle-specific expression of PRKAG3 is consistent with the fact that RN- animals show high glycogen content in skeletal muscle but not in liver. The R200Q mutation was found in all RN- animals but not in any rn+ animals from Hampshire or other breeds, supporting the assumption that RN- originated in the Hampshire breed. Milan et al. (2000) found that AMPK activity in muscle extracts was about 3 times higher in normal rn+ pigs than in RN- pigs, both in the presence and absence of AMP. The distinct phenotype of the RN- mutation indicates that PRKAG3 plays a key role in the regulation of energy metabolism in skeletal muscle.