Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1997-08-20
2002-02-12
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S006120, C435S007100, C435S015000, C536S023200
Reexamination Certificate
active
06346406
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the identification of a new superfamily of eukaryotic protein kinases and the use of one member of this superfamily, elongation factor-2 kinase (eEF-2 kinase), in assays to screen for specific inhibitors. Specific inhibitors of the eEF-2 kinase may be potent therapeutics for amelioration of malignant transformation. Additionally, sequences complementary to eEF-2 kinase may have therapeutic efficacy as antisense drugs or be used in gene therapy. Specifically, the invention relates to assays developed using the recombinant eEF-2 kinase to screen for inhibitors of phosphorylation of a peptide derived from the myosin heavy chain (MHC) protein.
BACKGROUND OF THE INVENTION
Protein phosphorylation plays a critical role in many cellular processes (Krebs (1994)
Trends Biochem. Sci
. 19:439; Hanks and Hunter, (1996)
FASEB J
. 9:576-596; Hardie and Hanks, (1995)
The Protein Kinase Facts Book
(Academic, London)). There are two well-characterized superfamilies of protein kinases, with most of the protein kinases belonging to the serine/threonine/tyrosine kinase superfamily (Hanks and Hunter, (1996); Hardie and Hanks, (1995)). The characterization of several hundred members of this superfamily revealed that they all share a similar structural organization of their catalytic domains which consist of twelve conserved subdomains (Hanks and Hunter, (1996); Hardie and Hanks, (1995)). The other superfamily is referred to as the histidine kinase superfamily and is involved in the prokaryotic two-component signal transduction system, acting as sensor components (Stock et al., (1989)
Microbiol. Rev
. 53:450-490; Parkinson and Kofoid, (1992)
Annu. Rev. Genet
. 26:71-112; Swanson, et al., (1994)
Trends Biochem. Sci
. 19:485-490). Recently, eukaryotic members of this superfamily have also been described (Chang et al., (1993)
Science
263:539-544; Ota and Varshavsky, (1993)
Science
262:566-569; Maeda et al., (1994)
Nature
369:242-245). Mitochondrial protein kinases have also recently been described that show structural homology to the histidine kinases, but phosphorylate their substrates on serine (Popov et al., (1992)
J. Biol. Chem
. 267:13127-13130; Popov et al., (1993)
J. Biol. Chem
. 268:26602-22606). Finally, several new protein kinases have been reported that show a lack of homology with either of the kinase superfamilies (Maru and Witte, (1991)
Cell
67:459-468; Beeler et al., (1994)
Mol. Cell. Biol
. 14:982-988; Dikstein et al., (1996) Cell 84:781-790; Futey et al., (1995)
J. Biol. Chem
. 270:523-529; Eichenger et al., (1996)
EMBO J
. 15:5547-5556). However, these protein kinases are viewed as an exception to the general rule as they have yet to be fully characterized.
The cloning and sequencing of the extensively characterized eukaryotic elongation factor-2 kinase (eEF-2 kinase) from a variety of eukaryotic organisms has now revealed the existence of a novel class of protein kinases (Ryazanov et al., (1997)
Proc. Natl. Acad. Sci., USA
94:4884-4889). eEF-2 kinase, previously known as Ca
2+
/calmodulin-dependent protein kinase III, is highly specific for phosphorylation of elongation factor-2 (eEF-2), an abundant cytoplasmic protein that catalyzes the movement of the ribosome along mRNA during translation in eukaryotic cells (reviewed in Ryazanov and Spirin, (1993) In
Translational Regulation of Gene Expression
(Plenum, New York) Vol. 2, pp. 433-455; Nairn and Palfrey, (1996) In
Translational Control
(CSHL Press, New York) pp. 295-318). All mammalian tissues, and various invertebrate organisms, exhibit eEF-2 kinase activity (Abdelmajid et al., (1993)
Int. J. Dev. Biol
. 37:279-290). eEF-2 kinase catalyzes the phosphorylation of eEF-2 at two highly conserved threonine residues located within a GTP-binding domain (Ryazanov and Spirin, (1993) In
Translational Regulation of Gene Expression
(Plenum, New York) Vol. 2, pp. 433-455; Nairn and Palfrey, (1996) In
Translational Control
(CSHL Press, New York) pp. 295-318). When eEF-2 is phosphorylated, it becomes inactive with respect to protein synthesis (Ryazanov et al., (1988)
Nature
334:170-173). Since eEF-2 phosphorylation is dependent on Ca
2+
and calmodulin, eEF-2 kinase plays a pivotal role in modulating the protein synthesis rate in response to changes in intracellular calcium concentration. Phosphorylation of eEF-2 has also been linked to the regulation of cell cycle progression. For example, transient phosphorylation of eEF-2 occurs during the mitogenic stimulation of quiescent cells (Palfrey et al., (1987)
J. Biol. Chem
. 262:9785-9792) and during mitosis (Celis et al., (1990)
Proc. Natl. Acad. Sci., USA
87:4231-4235). In addition, changes in the level of eEF-2 kinase activity is associated with a host of cellular processes such as cellular differentiation (End et al., (1982)
J. Biol. Chem
. 257:9223-9225; Koizumi et al., (1989)
FEBS Lett
. 253:55-58; Brady et al., (1990)
J. Neurochem
. 54:1034-1039), oogenesis (Severinov et al., (1990)
New Biol
. 2: 887-893), and malignant transformation (Bagaglio et al., (1993)
Cancer Res
. 53:2260-2264).
The sequence eEP-2 kinase appears to have no homology to either the Ca
2+
/calmodulin-dependent protein kinases or to any members of the known protein kinase superfamilies (Ryazanov et al., (1997)
Proc. Natl. Acad. Sci., USA
94:4884-4889). However, the recently described myosin heavy chain kinase A (MHCK A) from Dictyostelium (Futey et al., (1995)
J. Biol. Chem
. 270:523-529) shows a great deal of homology with eEF-2 kinase. These two kinases define a novel class of protein kinases that may represent a new superfamily.
Evidence for MHCK and eEF-2 kinase forming the core of a new superfamily is as follows. MHCK A from Dictyostelium, has a demonstrated role in the regulation of myosin assembly (Futey et al., (1995)
J. Biol. Chem
. 270:523-529; Côté et al., (1997)
J. Biol. Chem
. 272:6846-6849). eEF-2 kinase is a ubiquitous Ca
2+
/calmodulin-dependant protein kinase involved in the regulation of protein synthesis by Ca
2+
(Redpath et al., (1996)
J. Biol. Chem
271:17547-17554; Ryazanov et al., (1997)
Proc. Natl. Acad. Sci., USA
94:4884-4889). Both MHCK A and eEF-2 kinase display no homology to any of the known protein kinases, but are strikingly similar to each other; amino acid sequences of their catalytic domains are 40% identical. Another protein kinase homologous to MHCK A and eEF-2 kinase has recently been identified in Dictyostelium (Clancy et al., (1997)
J. Biol. Chem
. 272:11812-11815), and an expressed sequence tag (EST) sequence, with a high degree of similarity to the catalytic domain common to both MHCK A and eEF-2 kinase, has been deposited in GenBank (clone FC-AN09/accession #C22986). An amino acid sequence alignment of the catalytic domains of these new protein kinases is shown in FIG.
1
A. These kinases have a catalytic domain of approximately 200 amino acids which can be subdivided into seven conserved subdomains. Subdomains V, VI, and VII have a predicted &bgr;-sheet structure and are presumably involved in ATP-binding, while subdomains I through IV may be involved in substrate binding and catalysis. These new protein kinases have no homology to the members of the eukaryotic serine/threonine/tyrosine protein kinase superfamily with the exception of the GXGXXG motif in subdomain VI which is present in many ATP-binding proteins. Thus, MHCK A, eEF-2 kinase, and related protein kinases may represent a new superfamily. Evolutionary analysis of these new kinases (
FIG. 1B
) reveals that they can be subdivided into 2 families: the eEF-2 kinase family which includes eEF-2 kinases from different organisms, and the MHCK family which includes MHCK A, MHCK B and FC-AN09. These two families appear to have split more than a billion years ago.
An interesting question is why does nature employ these unusual kinases to phosphorylate eEF-2 and myosin heavy chains? Perhaps the answer is related to the secondary structure of the phosphorylation sites. As was originally re
Hait William N.
Pavur Karen S.
Ryazanov Alexey G.
Dietzel Christine E.
Klauber & Jackson
Moore William W.
University of Medicine & Dentistry of New Jersey
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