Nucleic acid encoding amp-activated protein kinase

Details Nucleic acid encoding amp-activated protein kinase Nucleic acid encoding amp-activated protein kinase

1996-03-06

2001-07-10

Carlson, Karen Cochrane (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S069100, C435S183000, C530S325000, C530S300000, C530S350000, C530S387100, C536S023200, C536S023400

Reexamination Certificate

active

06258547

ABSTRACT:

The key enzymes regulating fatty acid and cholesterol biosynthesis in the body are acetyl coA carboxylase (ACC) and HMG CoA reductase (HMG-R). The release of fatty acids and cholesterol from intracellular stores of triglycerides and cholesterol esters is regulated by hormone sensitive lipase (HSL). Regulation of these enzymes is multivalent eg feedback inhibition, transcriptional and translational control. More recently, reversible phosphorylation has also been discovered as a mechanism for the acute regulation of at least ACC and HMG-R, and possibly HSL. Work over the past few years has identified the key kinase that regulates the activities of these enzymes.
AMP protein kinase, which is itself regulated allosterically by 5′-AMP and reversible phosphorylation (by a distinct kinase—kinase), can phosphorylate and inactivate ACC and HMG-R (& possibly HSL). Although agents which inhibit HMG-R, such as members of the statin family of compounds, have found great utility in the clinic for the treatment of hypercholesterolaemia, there are no acceptable agents for the treatment of combined hyperlipidaemia ie elevated fatty acids & elevated cholesterol levels; elevated fatty acids are increasingly being recognised as important in numerous disease states.
Carling et al (Eur. J. Biochem., 1989, 186, 129-136) describe the purification of an AMP protein kinase from rat liver. However, all efforts to clone a mammalian AMP protein kinase appear to have been unsuccessful.
According to a first aspect of the present invention we now provide a cDNA encoding a mammalian AMP protein kinase and convenient fragments thereof. The cDNA is conveniently as set out in Table 1 and a restriction map indicating convenient fragments thereof is set out in Table 3.
In a further aspect of the present invention we provide a recombinant mammalian AMP protein kinase and convenient fragments thereof. This may for example be obtained by expression of the cDNA as set out in SEQ ID NO: 24 or convenient fragments thereof as indicated in FIG.
9
. Convenient peptide sequence which may be obtained is set out in FIG.
8
. Methods for cDNA expression will be apparent to the molecular biologist of ordinary skill and include those set out in the Maniatis cloning manual (Molecular Cloning: A Laboratory Manual—2nd Edition (1989)—J. Sambrook, E. F. Fritsch & Maniatis), and in Current Protocols in Molecular Biology—1987—P. Sharp (Ed.). Particular expression systems include the mouse erythroleukaemia (mel) cell expression system claimed in WO-89/01517 (Grosveld) and, more particularly, as claimed in WO-92-11380 (Hollis et al). An alternative expression system is the Baculovirus Expression System (Clontech)—A. Prokop et al, Recombinant DNA Technology & Applications, 1991, 97-152.
The recombinant mammalian AMP protein kinase of the present invention is used to identify agents which modulate the action of this kinase. Such agents are desirable since, for example, they can be used to reduce the biosynthesis of cholesterol and fatty acids. They may also be used to inhibit the release of these from intracellular stores by HSL.
Therefore in a further aspect of the present invention we provide a method for the identification of an agent which increases activation of AMP protein kinase, which method comprises contacting a potential agent with a recombinant mammalian AMP protein kinase and identifying any increase in AMP protein kinase activation over that found in the absence of the agent. Any convenient assay format may be used. In particular a peptide, for example containing a serine residue exclusively phosphorylate by AMP protein kinase is incubated in the presence of a preparation of AMP protein kinase and a radiolabel such as gamma 33-P. The reaction is allowed to proceed for a period of about 1 hour and is conveniently terminated by the addition of acid. The phosphorylated peptide is conveniently separated from unicorporated radiolabel by binding to a charged membrane followed by washing. The degree of phosphorylation of the peptide is a measure of the activity/activation of the AMP protein kinase.
In a further aspect of the invention we provide a method for the identification of an agent which increases the level of AMP protein kinase expression, which method comprises contacting a potential agent with a cell expressing a mammalian AMP protein kinase and identifying any increase in the level of AMP protein kinase expression over that found in the absence of the agent. This method may involve the use of any convenient in vitro assay. In general, a cell expressing the mammalian AMP protein kinase gene is contacted with an agent capable of transcriptionally modulating expression of the gene, thereby affecting the level of protein encoded by the gene which is expressed by the cell. Convenient procedures are described in WO90/01379 (Oncogene). An increase in transcription may be monitored by blotting techniques using probes derived from the cDNA encoding the AMP protein kinase, or by PCR using primer sequences derived from the cDNA. Protein expression levels can be conveniently monitored by the use of antibodies and enzyme activity measured as described above.
The cDNA of the invention encoding a mammalian AMP protein kinase, or any convenient fragment thereof, may be used to provide DNA probes. A convenient probe comprises the full cDNA sequence of a mammalian AMP protein kinase, for example the cDNA sequences set out in SEQ ID NO: 24. The molecular biologist of ordinary skill will be aware that the above DNA probes (or RNA probes derived therefrom) may be use in a number of procedures. These include the identification and cloning of homologous mammalian and non-mammalian cDNAs including the associated kinase—kinase. Such homologous cDNAs represent further independent aspects of the invention. They may be cloned into vectors (such as those commercially available) in order to produce useful fusion proteins or cloned into expression vectors to construct high level expressing cell lines. They may also be used in gene cloning studies in order to analyse a number of regulatory elements including promoters, enhancers and introns. In addition they may be used to investigate gene expression in vivo. They may also be used in the preparation of transgenic animals, such as mice or rats.
As outlined earlier above the recombinant mammalian AMP protein kinase of the invention, and convenient fragments thereof, may be used to identify agents which cause increased enzyme activation. It may be used in molecular modelling and X-ray crystallography studies. In addition it can be used to map the site(s) phosphorylated by the putative kinase—kinase (which causes activation of AMP protein kinase) and de-phosphorylated by a kinase-phosphatase (inactivated AMP protein kinase). This is conveniently achieved by working out sequentially (i) the number of sites phosphorylated on the protein (ii) which site(s) are phosphorylated by which protein kinase (in vitro) and (iii) which kinase(s) are responsible for phosphorylation in vivo. Based on the data derived from the above types of experiments, and AMP protein kinase peptide substrate specific for the kinase—kinase and kinase-phosphatase may be developed to allow specific assays for the latter enzymes.
The recombinant mammalian AMP protein kinase of the invention, and convenient fragments thereof, may also be used to raise antibodies. Such antibodies have a number of uses which will be evident to the molecular biologist of ordinary skill. These include (i) monitoring protein expression in native cells and clones which express recombinant AMP protein kinase, (ii) the development of assays to measure kinase-kinase and kinase phosphatase and (iii) the precipitation of AMP protein kinase and other proteins which associate with AMP protein kinase leading to identification of these proteins.
We have found that AMPK shows 46% sequence identity to the protein encoded by the yeast SNF 1 gene, the product of which is essential for release from glucose repression. Whilst we do not wish to be bound by theoretical cons

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