Human MEKK proteins, corresponding nucleic acid molecules,...

Details Human MEKK proteins, corresponding nucleic acid molecules,... Human MEKK proteins, corresponding nucleic acid molecules,...

2000-03-06

2001-11-06

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S252300, C435S320100, C435S325000, C435S006120, C536S023200

Reexamination Certificate

active

06312934

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to isolated nucleic acid molecules encoding MEKK proteins, substantially pure MEKK proteins, and products and methods for regulating signal transduction in a cell.
BACKGROUND OF THE INVENTION
Mitogen-activated protein kinase (MAPKs) (also called extracellular signal-regulated kinases or ERKs) are rapidly activated in response to ligand binding by both growth factor receptors that are tyrosine kinases (such as the epidermal growth factor (EGF) receptor) and receptors that are coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) such as the thrombin receptor. In addition, receptors like the T cell receptor (TCR) and B cell receptor (BCR) are non-covalently associated with src family tyrosine kinases which activate MAPK pathways. Specific cytokines such as tumor necrosis factor (TNF&agr;) can also regulate MAPK pathways. The MAPKs appear to integrate multiple intracellular signals transmitted by various second messengers. MAPKs phosphorylate and regulate the activity of enzymes and transcription factors including the EGF receptor, Rsk 90, phospholipase A
2
, c-Myc, c-Jun and Elk-1/TCF. Although the rapid activation of MAPKs by receptors that are tyrosine kinases is dependent on Ras, G protein-mediated activation of MAPK appears to occur through pathways dependent and independent of Ras.
The MAPKs are activated by phosphorylation on both a threonine and tyrosine by dual specificity kinases, MAPK/ERK kinases (MEKs) which are, in turn, activated by serine/threonine phosphorylation MAPK kinase kinases (MKKKs or MEKKs). At present, at least four MEKKs have been identified. The four MEKK proteins range from 69.5-185 kDa in size, having their kinase domains in the carboxy-terminal end of the protein and their catalytic domains in the amino-terminal end of the protein. Murine MEKK1 was cloned initially on the basis of its homology with the STE11 and Byr2 kinases from yeast (Lange-Carter et al. (1993)
Science
260:315-319; Xu et al. (1996)
Proc. Natl. Acad. Sci. USA
93:5291-5295; and Blank et al. (1996)
J. Biol. Chem
. 271:5361-5368). Murine MEKK2 and MEKK3 were subsequently cloned and found to have 94% homology in their kinase domains as well as 65% homology within their catalytic domains. Blank et al., supra. The cloning of murine MEKK4 revealed approximately 55% homology to the kinase domains of MEKKs 1, 2, and 3 whereas the amino-terminal region of MEKK4 has little sequence homology to the other MEKK family members. Gerwin et al. (1997)
J. Biol. Chem
. 272:8288-8295. MEKK1 and MEKK4, but not MEKK2 and MEKK3, bind to the low molecular weight GTP-binding proteins Cdc42 and Rac. Furthermore, MEKK1 also binds to Ras in a GTP-dependent manner (Russell et al. (1996)
J. Biol. Chem
. 11757-11760) and Ras activity is required for EGF-mediated stimulation of MEKK1 activity (Lange-Carter and Johnson (1994)
Science
265:1458-1461). In addition to growth factor receptor tyrosine kinases (i.e. EGF receptor), the TNF receptor, the Fc&egr;R1 in mast cells Ishizuka et al. et al. (1996)
J. Biol. Chem
. 271:12762-12766) and the N-formyl methionyl leucine peptide receptor in neutrophils have been shown to activate MEKK1. EGF and TNF also activate MEKK3 and it also appears that the other MEKK proteins are regulated by tyrosine kinase receptors but the intermediate components and effector molecules leading to their activation are poorly understood.
The cellular effects of MEKK1 are quite diverse and can be classified as being either JNK-dependent or JNK-independent. For example, MEKK1 can mediate activation of ERKI and ERK2 and, by a yet undefined mechanism, activation of the c-Myc transcription factor independent of JNK activity (Lassignal-Johnson et al. (1996)
J. Biol. Chem
. 271:3229-3237 and Lange-Carter et al. (1993)
Science
260:315-319). Alternatively, MEKK1 may or may not require JNK activity for activation of IKB kinase which leads to NK&kgr;B activation (Liu et al. (1996)
Cell
87:565-576 and Meyer et al. (1996)
J. Biol. Chem
. 271 :8971-8976). Furthermore, depending upon the cell type, MEKK1, but not MEKK2, 3 or 4, has been shown to mediate apoptosis by both JNK-dependent and JNK-independent mechanisms (Xia et al. (1995)
Science
270:1326-1331 and Lassignal-Johnson et al. (1996)
J. Biol. Chem
. 271:3229-3237).
Given the important role of members of the MAPK signal transduction cascade, in particular the MEKK signal transduction molecules, in regulating mammalian cellular processes ranging from cellular proliferation and differation to cellular apoptosis, there exists a need for identifying human MEKK nucleic acid and protein molecules as well as for modulators of such molecules for use in regulating a variety of human cellular responses.
SUMMARY OF THE INVENTION
This invention provides human MEKK compositions. In particular, this invention provides isolated nucleic acid molecules encoding human MEKK1, human MEKK2, and human MEKK3. The invention further provides isolated human MEKK1, human MEKK2, and human MEKK3 proteins. Because the MEKK compositions of the invention are human-derived, they function optimally in human cells (compared with non-human MEKK compositions) and do not stimulate an immune response in humans.
One aspect of the invention pertains to an isolated nucleic acid molecule having a nucleotide sequence which encodes a human MEKK protein. In a preferred embodiment, the nucleic acid molecule has the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. In other embodiments, the nucleic acid molecule has at least 90-91% nucleotide identity, more preferably 92-93% nucleotide identity, more preferably 94-95% nucleotide identity, more preferably 96-97% nucleotide identity, more preferably 98-99% nucleotide identity, and even more preferably 99.5% nucleotide identity with the nucleotide sequence SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.
The isolated nucleic acid molecules of the invention encoding human MEKK proteins can be incorporated into a vector, such as an expression vector, and this vector can be introduced into a host cell. The invention also provides a method for producing a human MEKK protein by culturing a host cell of the invention (carrying a huMEKK1, huMEKK2, or huMEKK3 expression vector) in a suitable medium until a human MEKK protein is produced. The method can further involve isolating the human MEKK protein from the medium or the host cell.
Another aspect of the invention pertains to an isolated human MEKK proteins. Preferably, the human MEKK protein has the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. In other embodiments, the protein has at least 90-91% amino acid identity, more preferably 92-93% amino identity, more preferably 94-95% amino identity, more preferably 96-97% amino identity, more preferably 98-99% amino identity, and even more preferably 99.5% amino acid identity with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
Fusion proteins, including a human MEKK protein operatively linked to a polypeptide other than human MEKK, are also encompassed by the invention, as well as antibodies that specifically bind a human MEKK protein. The antibodies can be, for example, polyclonal antibodies or monoclonal antibodies. In one embodiment, the antibodies are coupled to a detectable substance.
Another aspect of the invention pertains to a nonhuman transgenic animal that contains cells carrying a transgene encoding a human MEKK protein.
Yet another aspect of the invention pertains to a method for detecting the presence of human MEKK in a biological sample. The method involves contacting the biological sample with an agent capable of detecting an indicator of human MEKK activity such that the presence of human MEKK is detected in the biological sample. The invention also provides a method for modulating human MEKK activity in a cell which involves contacting the cell with an agent that modulates human MEKK activity such that human MEKK activity in the cell is modulated.
Still another aspect of the invention pertains to methods for identif

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