Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1996-04-05
2001-12-25
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C536S023100, C536S024310
Reexamination Certificate
active
06333170
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 (TCR) and B cell (BCR) receptors are non-covalently associated with src family tyrosine kinases which activate MAPK pathways. Specific cytokines like 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.
Complementation analysis of the pheromone-induced signaling pathway in yeast has defined a protein kinase system that controls the activity of Spk1 and Fus3-Kss1, the
Schizosaccharomyces pombe
and
Saccharomyces cerevisiae
homologs of MAPK (see for example, B. R. Cairns et al.,
Genes and Dev.
6, 1305 (1992); B. J. Stevenson et al.,
Genes and Dev.
6, 1293 (1992); S. A. Nadin-Davis et al.,
EMBO J
7, 985 (1988); Y. Wang et al.,
Mol. Cell. Biol.
11, 3554 (1991). In
S. cerevisiae
, the protein kinase Ste7 is the upstream regulator of Fus3-Kss1 activity; the protein kinase Ste11 regulates Ste7. The
S. pombe
gene products Byr1 and Byr2 are homologous to Ste7 and Ste11, respectively. The MEK (MAPK Kinase or ERK Kinase) or MKK (MAP Kinase kinase) enzymes are similar in sequence to Ste7 and Byr1. The MEKs phosphorylate MAPKs on both tyrosine and threonine residues which results in activation of MAPK. The mammalian serine-threonine protein kinase Raf phosphorylates and activates MEK, which leads to activation of MAPK. Raf is activated in response to growth factor receptor tyrosine kinase activity and therefore Raf may activate MAPK in response to stimulation of membrane-associated tyrosine kinases. Raf is unrelated in sequence to Ste11 and Byr2. Thus, Raf may represent a divergence in mammalian cells from the pheromone-responsive protein kinase system defined in yeast. Cell and receptor specific differences in the regulation of MAPKs suggest that other Raf independent regulators of mammalian MEKs exist.
Certain biological functions, such as growth and differentiation, are tightly regulated by signal transduction pathways within cells. Signal transduction pathways maintain the balanced steady state functioning of a cell. Disease states can arise when signal transduction in a cell breaks down, thereby removing the tight control that typically exists over cellular functions. For example, tumors develop when regulation of cell growth is disrupted enabling a clone of cells to expand indefinitely. Because signal transduction networks regulate a multitude of cellular functions depending upon the cell type, a wide variety of diseases can result from abnormalities in such networks. Devastating diseases such as cancer, autoimmune diseases, allergic reactions, inflammation, neurological disorders and hormone-related diseases can result from abnormal signal transduction.
SUMMARY OF THE INVENTION
The present invention relates to a substantially pure MEKK protein capable of regulating a MEK kinase dependent pathway. In certain embodiments a MEK kinase comprises a catalytic domain and is capable of phosphorylating MKK proteins. In preferred embidiments the MEKK substrate is seleted from the group of MAP kinase kinases consisting of MEKK1, MKK2, (also called MEK1 and MEK2 respectively) MKK3, or MKK4 (also called JNKK1 and JNKK2 or SEK respectively). The present invention includes a substantially pure MEKK protein capable of regulating signals initiated from a growth factor receptor on the surface of a cell by regulating the activity of MAPK protein. Exemplary MAP kinases include p42, p44, ERK1, ERK2, JNK1, JNK2, or p38 SAPK. In preferred embodiments a MEK kinase can activate at least one of the group c myc, cJun, cPLA2, Rsk 90, TCF, Elk-1, or ATF-2.
In certain embodiments the MEKK protein of the present invention is regulates the activity of a MAPK protein independently of Raf. In preferred embodiments the MEKK proteins described herein are capable of binding members of the Ras superfamily. Exemplary polypeptides which bind to MEKK proteins include Ras, Rac/Cdc42, or Rho.
In particular, the substantially pure MEKK proteins of the present invention comprise at least a portion of an amino acid sequence shown in one of SEQ ID NOS:2, 4, 6, 8, 10, 12, or 14. In other embodiments, proteins at least 50% homologous, at least 75% homologous, preferably at least 85% homologous, or more preferably 95% homologous to one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, or 14 are also contemplated.
In certain embodiments MEKK proteins have homology to the kinase catalytic domain of one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, or 14. In other embodiments proteins having at least 50% homology, at least 75% homology, preferably at least 85% homology, or more preferably at least 95% homology to the kinase catalytic domain of one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, or 14 are contemplated. In more preferred embodiements the kinase domain of a MEKK protein is capable of phosphorylating a MAP kinase kinase protein and binding to a member of the ras superfamily, such as ras or rac or cdc42 protein.
In another embodiment the MEKK protein of the present invention comprises a NH2 regulatory domain represented in one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, or 14. In other embodiments MEKK proteins which comprise regions of at least 50% homology, at least 75% homology, preferrably 85% homology, or more preferably at least 95% homology to the NH2 regulatory domain of one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, or 14 are contemplated.
In a further embodiment MEKK proteins which have molecular weights ranging from 60 to 190 are contemplated. Preferred molecular weights are 98 kD for MEKK1, 69.5 kD for MEKK2, 71 kD for MEKK3, and 95-98 kD for MEKK 4. In other embodiments MEKK 4 migrates with an apparent molecular weight of 185 kD.
MEKK proteins of the present invention lack an SH2 or SH3 domain. In preferred embodiments exemplary MEKK proteins comprise a proline rich SH3 binding motif. In certain embodiments, MEKK proteins of the instant invention comprise a Pleckstrin homology domain.
In a particularly preferred embodiment, exemplary MEKK proteins can competitively inhibit the activity of a MEKK designated in one or more of SEQ ID NOS: 2, 4, 6, 8, 10, or 12, or 14.
Fragments of MEKK proteins are also contemplated by the present invention. In preferred embodiments exemplary MEKK proteins lack a MEKK regulatory domain. In particularly preferred embodiments MEKK protein fragments lack the scrine/threonine rich regulatory domain shown in one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, or 14. In another embodiment the fragement of a MEKK protein lacks the serine/threonine kinase domain of a MEKK protein. In preferred embodiments MEKK protein fragments lack the serine/threonine kinase domain shown in one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, or 14.
In another embodiment the MEKK protein of the present invention is a fusion protein further comprising, in addition to the MEKK polypeptide, a second polypeptide sequence having an amino acid sequence unrelated to MEKK polypeptide sequence. In a preferred embodiment the fusion protein includes as a second polypeptide sequence, a polypeptide which functions as a detectable label for detec
Basi Nirmal S.
DeConti, Jr. Esq. Guilio A.
Kemmerer Elizabeth
Lahive & Cockfield LLP
Lauro, Esq. Peter C.
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