MEKK1 molecules and uses thereof

Details MEKK1 molecules and uses thereof MEKK1 molecules and uses thereof

2000-10-27

2004-11-16

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S252300, C435S320100, C435S325000, C536S023200

Reexamination Certificate

active

06818427

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the identification of isolated, full-length human MEKK1 nucleic acid molecules encoding MEKK1 proteins, and methods of using the nucleic acid molecules and proteins.
BACKGROUND OF THE INVENTION
Controlling the state of phosphorylation is an important mechanism by which signaling molecules regulate the activity of other proteins. One common theme in such molecular signaling is the so-called kinase cascade, in which a linear series of kinases is activated by phosphorylation by upstream kinases. The mitogen-activated protein kinase (MAPK) pathway is one such example of a kinase cascade.
MAPKs are activated and phosphorylated by MAPK kinases (MKKs). The MKKs, in turn, are activated and phosphorylated by serine/threonine kinases (MKKKs), which themselves may be activated and phosphorylated by MKKK kinases (MKKKKs). There are currently over ten different groups of kinases covering more than twenty-two different genes that act upstream of one another and regulate the MKKs. One family, the MAPK/ERK kinase kinases (MEKKs) directly activate and phosphorylate specific MKKs. (Schlesinger et al.,
Front Biosci.
3:d1181-1186 (1998).)
MEKKs are activated by a number of diverse extracellular stimuli, indicating that not only can these molecules affect a wide variety of downstream activity, but they can also react to a diverse array of extracellular stimuli. For example, both EGF receptor stimulation and TNF a lead to an increase in MEKK1 activity. (Lange-Carter et al.,
Science
265:1458-1461 (1994); Winston et al.,
Proc. Natl. Acad. Sci.,
92:1614-1618 (1995); Ishizuka et al.,
J. Biol. Chem.
271:12762-12766 (1996); Kaga et al.,
J. Immunol.
160:4182-4189 (1998).) MEKK1 is also activated in response to DNA damaging stresses such as UV irradiation, etoposide, cisplatin, and mitomycin C. (Widmann et al.,
Mol Cell. Biol.
18:2416-2429 (1998); Cardone et al.,
Cell
90:315-323 (1997).)
To date, six different MEKK genes have been at least partially cloned in mammalian cells. (Lange-Carter et al.,
Science
260:315-319 (1993); Blank et al.,
J. Biol. Chem.
271:5361-5368 (1996); Gerwins et al.,
J. Biol. Chem.
272:8288-8295 (1997); Wang et al.,
J. Biol. Chem
271:31607-31611 (1996); Wang et al.,
Biochem. Biophys. Res. Commun.
253:33-37 (1998).) There are a number of functional motifs found within the N-terminal regulatory domains of, at least, MEKK1 and MEKK4. Both molecules contain putative pleckstrin homology domains. Pleckstrin domains associate with polyphosphoinositides and mediate localization to specific regions of the plasma membrane. MEKK1 and MEKK4 also contain proline rich regions at the N-terminus, which may be of functional significance. Proline rich regions have been shown to be important for binding proteins that contain Src homology 3 (SH3) domains. Moreover, with regard to MEKK1, 14-3-3 proteins bind at the N-terminal regulatory domain. Although 14-3-3 association does not appear to dramatically affect MEKK activity, 14-3-3 proteins are important for MEKK regulation by mediating interactions with other regulatory proteins and for controlling subcellular localization of these kinases.
MEKK1 is important in regulating cell survival and apoptosis. MEKK1 is a substrate for caspases, a family of proteases required for apoptosis. The apoptotic signaling appears to be dependent on cleavage of MEKK1 and subsequent caspase activation, as cleavage resistant mutants do not induce apoptosis. Thus, MEKK1 plays a critical role in regulating cell survival and death, acting as a molecular switch when cleaved by caspases. Cleavage by a caspase changes MEKK1 from a survival promoting kinase to an effector of cell death.
MEKK1 activates both the Activator Protein-1 (AP-1) stress response pathway and the NF&kgr;B pathway. The transcription factor AP-1 is a critical regulator of T-cell activation, cytokine production, including IL-2, IL-3, and GM-CSF, and the production of metalloproteinases. (Gottschalf et al.,
J. Exp. Med.
178:1681 (1993); Want et al.,
J. Mol. Cell. Biol.
14:11153 (1994); Rao,
Immunol. Today
15:274 (1994); Angel et al.,
Biochem. Biophys. Acta.
1072:129 (1991).) With regard to cytokine regulation, AP-1 mediates positive transactivation independently or in association with NF-AT (Nolan,
Cell
77:795 (1994)). AP-1 activity is induced by many stimuli, including the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA), growth factors, cytokines, T-cell activators, neurotransmitters, and UV irradiation. AP-1 is composed of dimers of different members of the Fos and Jun family of proteins. AP-1 activity is regulated at the level of both C-Jun and C-Fos transcription and by post-translational modification of their protein products by phosphorylation and dephosphorylation.
Moreover, independent of its MAPK activity, MEKK1 also plays another role in regulating transcription factor NF&kgr;B, which is a dimer maintained in the cytoplasm via an inhibitory regulatory subunit, I&kgr;B. (Lee et al.,
Cell
88:213 (1997).) Upon stimulation with specific cytokines or environmental stresses, I&kgr;B is phosphorylated by I&kgr;B kinase which induces proteolytic degradation of I&kgr;B, thereby releasing NF&kgr;B to translocate to the nucleus effecting changes in transcription. (PCT Publication No. WO 97/35014.) Over expression of MEKK1 activates NF&kgr;B.
The two transcription factors, NF&kgr;B and AP1, have been shown to regulate the production of many proinflammatory cytokines and related proteins that are elevated in immunoinflammatory diseases. These transcription factors regulate IL-1, IL-2, TNF &agr;, IL-6, and IL-8 levels in a variety of cell types. NF&kgr;B and other related transcription factor complexes are involved in the rapid induction of genes whose products function in protective and proliferative responses upon exposure of cells to external stimuli. Similarly, AP-1 has a significant role in the regulation of IL-2 transcription during T-cell activation. Thus, the role of NF&kgr;B and AP-1 is to act as a transducer of certain stimuli that lead to immune, inflammatory, and acute phase responses and, when overactivated, can lead to a disease state (Suto et al.,
Current Pharm. Design
3:515-528 (1997). No known antiinflammatory or autoimmune drugs have been specifically developed clinically as inhibitors of NF&kgr;B or AP-1. Therefore, a critical need exists for new therapies to treat immunoinflammatory and autoimmune disorders.
Prior to the instant invention, cloning efforts to obtain the full-length human MEKK1 gene have been unsuccessful for several reasons. Earlier efforts by others failed to identify the full-length cDNA. In some instances, the libraries that were screened were 5′-stressed and would not have expressed a full-length coding region. Specifically, the 5′ region of the gene proved difficult to clone for reasons that were, until now, unclear. Other attempts using reverse PCR methodology were also unsuccessful.
Nevertheless, in order to completely understand MEKK1 biology and to develop potential regulators or modulators of MEKK1, the full-length MEKK1 nucleic acid molecule had to be cloned and isolated. There is a need, 9 therefore, to identify and clone the full-length MEKK1 nucleic acid to further understand and exploit the role of MEKK1.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery of a novel fill-length human gene referred to herein as “MEKK1”. The polynucleotide sequence of a cDNA encoding a MEKK1 polypeptide is shown in SEQ ID NO:1, and the amino acid sequence of a MEKK1 polypeptide is shoen in SEQ ID NO:2. In addition, the polynucleotide sequence of the coding region is from nucleotide 7 to nucleotide 4545 of SEQ ID NO:1.
Accordingly, in a first aspect, the invention features a full-length nucleic acid molecule which encodes a MEKK1 protein or polypeptide, e.g., a biologically active portion of the MEKK1 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID

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