Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Patent
1998-10-07
2000-09-26
Prouty, Rebecca E.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
4352523, 4353201, 435325, 536 232, C12N 912, C12N 120, C12N 1500, C12N 500, C07H 2104
Patent
active
061241253
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The present invention relates to novel AMP protein kinase subunits, to polynucleotides encoding these subunit proteins and to antibodies which bind to these subunits.
The 5'-AMP-activated protein kinase (AMPK) was initially identified as a protein kinase regulating HMG-CoA reductase (Ferrer et al. (1985) Biochem. Biophys. Res. Commun. 132, 497-504). Subsequently, the AMPK was shown to phosphorylate hepatic acetyl-CoA carboxylase (Carling et al. (1987) FEBS Lett. 223, 217-222) and adipose hormone-sensitive lipase (Garton et al. (1989) Eur. J. Biochem. 179, 249-254). The AMPK is therefore thought to play a key regulatory role in the synthesis of fatty acids and cholesterol.
The AMPK is believed to act as a metabolic stress-sensing protein kinase switching off biosynthetic pathways when cellular ATP levels are deleted and when 5'-AMP rises in response to fuel limitation and/or hypoxia (Corton et al. (1994) Current Biology 4, 315-324). Partial amino acid sequencing of hepatic AMPK (Mitchelhill et al. (1994) J. Biol. Chem. 269, 2361-2364; Stapleton et al. (1994) J. Biol. Chem. 269, 29343-29346) revealed that it consists of 3 subunits, the catalytic subunit .alpha. (63 kDa), and two non-catalytic subunits, .beta. (40 kDa) and .gamma. (38 kDa).
The AMPK is a member of the yeast SNF1 protein kinase subfamily that includes protein kinases present in plants, nematodes and humans. The AMPK catalytic subunit, .alpha., has a strong sequence identity to the catalytic domain of the yeast protein kinase SNF1, which is involved in the induction of invertase (SUC2) under conditions of nutritional stress due to glucose starvation (Celenza, J. L. and Carlson, M. (1986) Science 233, 1175-1180). Both snf1p and the AMPK require phosphorylation by an activating protein kinase for full activity. The sequence relationship between snf1p and AMPK led to the finding that these enzymes share functional similarities, both phosphorylating and inactivating yeast acetyl-CoA carboxylase (Woods et al. (1994) J. Biol. Chem. 269, 19509-19516; Witters, L. A. and Watts, T. D. (1990) Biochem. Biophys. Res. Commun. 169, 369-376). The non-catalytic .beta. and .gamma. subunits of AMPK are also related to proteins that interact with snf1p; the .beta. subunit is related to the SIP1/ SIP2 /GAL83 family of transcription regulators and the .gamma. subunit to SNF4 (also called CAT-3) (Yang et al. (1994) EMBO J. 13, 5878-5886).
An isoform of the mammalian AMPK catalytic subunit has previously been cloned (Carling et al. (1994) J. Biol. Chem. 269, 11442-11448) and is referred to herein as AMPK .alpha..sub.2. The sequence of AMPK is disclosed in WO 94/28116. The AMPK .alpha..sub.2 does not complement SNF1 in yeast, indicating that their full range of functions are not identical.
A novel isoform of the mammalian AMPK catalytic subunit has now been identified and is referred to herein as AMPK .alpha..sub.1. In addition, full-length cDNAs for the mammalian AMPK .beta. and AMPK .gamma. subunits have now been cloned and polypeptides encoded thereby purified.
SUMMARY OF THE INVENTION
Accordingly, a first aspect of the present invention provides an isolated polynucleotide which encodes mammalian AMPK .alpha..sub.1 or a sequence which hybridizes thereto with the proviso that the sequence does not hybridize to mammalian AMPK .alpha..sub.2 as defined in Table 1 or Table 5 of WO 94/28116. In a preferred embodiment, the polynucleotide comprises SEQ ID NO: 44. Also provided are vectors comprising such a polynucleotide, a host cell transformed with such a vector and recombinant proteins encoded by such a polynucleotide.
In a second aspect, the present invention provides a method of producing mammalian AMPK .alpha..sub.1 which comprises culturing the cell including the polynucleotide of the first aspect of the present invention under conditions which allow expression of the polynucleotide encoding AMPK .alpha..sub.1 and recovering the expressed AMPK .alpha..sub.1.
In a third aspect, the present invention provides an oligonucleotide probe of
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Kemp Bruce E.
Mitchelhill Kenneth I.
Stapleton David I.
Witters Lee A.
Licata Law Offices of Jane Massey
Monshipouri M.
Prouty Rebecca E.
St. Vincent's Institute of Medical Research
Trustees of Dartmouth College
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