Identification and functional characterization of a novel...

Details Identification and functional characterization of a novel... Identification and functional characterization of a novel...

2001-05-29

2004-12-14

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S015000, C435S320100, C435S069100, C435S325000, C435S252300, C536S023200, C536S023100, C530S350000

Reexamination Certificate

active

06830909

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a novel S6 kinase (p70&bgr;
Sk6
), mutant variants thereof methods of making and using this S6 kinase, and related nucleic acids and Ski antibodies. The invention also relates to binding partners of the S6 kinase, methods of identifying the binding partners and antibodies thereto.
BACKGROUND OF THE INVENTION
The 40S ribosomal protein S6 is a component of the 40S subunit of eukaryotic ribosomes. The ribosomes are part of the cellular machinery responsible for translation of mRNA and protein synthesis. The S6 protein is phosphorylated in response to certain cellular signaling events such as hormone or growth factor induced cellular proliferation. p70 S6 kinase (p70
S6k
) is responsible for S6 phosphorylation and is believed to be the major physiological S6 kinase in mammalian cells (Proud, 1996
Trends Biochem. Sci.
21:181-185).
1. p70&agr; S6 Kinase
A. Structure and Function
The first p70 S6 kinase identified was the alpha (&agr;) form. The gene encoding the human p70&agr; S6 kinase (70
S6k
)was isolated in 1991 (Grove et al., 1991
Mol. Cell. Biol.
11:5541-5550). Other p70&agr; S6 kinase sequences have been described in
Mus musculus
(GenBank Accession No. SEG_AB015196S, AB015197, and AB015196),
Xenopus laevis
(GenBank Accession No. X66179), and rat (GenBank Accession No. M57428).
Two p70&agr; S6 kinase isoforms were identified: p70&agr;-I GenBank Accession No. M60724) and p70&agr;-II (GenBank Accession No. M60725). The two p70&agr; S6 kinase isoforms differ only in their amino tenmini by 23 amino acid residues resulting in a 70 kD protein and a 85 kD protein. The isoforms are referred to in the literature as p70
S6k
/p85
S6k
or p70&agr; S6 kinase. Both isoforms share similar activity towards ribosomal protein S6 in vitro but are expressed in different cells and tissues. The two isoforms are produced by two mRNA products and are not a result of post-translational modifications. They are serine/threonine kinases and are known to act on the substrate KKRNRTLSVA (SEQ ID No. 7) (Pai et al., 1994
Eur. J. Immunol.
24:2364-8; and Leighton et al., 1995
FEBS Letters
375:289-93).
The p70&agr; S6 kinase plays an important role in the progression of cells from G1 to S phase of the cell cycle and in the initiation of protein synthesis. Recently, p70&agr; S6 kinase has been demonstrated to regulate the translation of a class of mRNAs containing an oligopyrimidine tract in their 5′ untranslated region. This class of mRNAs, termed 5′TOP mRNAs, represent up to 20% of the a cell's total mRNA. Many of the proteins encoded by 5′TOP mRNAs are translational apparatus proteins and cell-cycle progression proteins.
The p70&agr; S6 kinase has four identified interdependent domains: (1) a catalytic domain, (2) a kinase extension domain, (3) a pseudosubstrate autoinhibitory domain, and (4) the N-terminal domain. The catalytic domain is located in the middle of the protein and is followed by the kinase extension domain, which is a unique feature for the PKA family. The pseudosubstrate autoinhibitory domain is also unique for the p70&agr; S6 kinase, not having been observed in any other known kinases. It possesses 5 phosphorylation sites which are responsible for the p70&agr; S6 kinase regulation. The N-terminal domain mediates the sensitivity for rapamycin, which strongly inhibits serum-induced phosphorylation and activation of the p70&agr; S6 kinase. This domain may also mediate the interaction with a yet unknown phosphatase.
B. Regulators and Cascades
Growth factors, such as insulin, and mitogens are known to activate in vivo p70&agr; S6 kinase (Alessi et al., 1998
Curr. Biol.
8:69-81). Heat shock also activates p70&agr; S6 kinase (Lin et al., 1997
J. Biol. Chem.
272:31196-31202). Certain drugs have been identified that regulate p70&agr; S6 kinase activity including: rapamycin, wortmannin, Ro31-8220, GF109203X, LY294002, phenylephrine (PE), PD098059, SQ20006, polymerized collagen, forskolin, interleukin-10 (IL-10), demethoxyviridin, phorbol 12-myristate 13-acetate (PMA), A23187, bombesin and antibodies which recognize the p70&agr; S6 kinase (Proud, 1996; Morreale et al., 1997
FEBS Letters
417:38-42; Kanda et al., 1997
J. Biol. Chem,
272:23347-23353; Boluyt et al., 1997
Circ Res.
81:176-186; Coolican et al, 1997
J. Biol. Chem.
272:6653-6662; Koyama et al., 1996 Cell 87: 1069-1078; Busca et al., 1996
J. Biol. Chem.
271:31824-31830; Crawley et al., 1996
J. Biol. Chem.
271:16357-16362; and Petritsch et al., 1995
Eur, J. Biochem.
230:431-8). The immunosuppressant rapamycin (Rap) is the most potent inhibitor of p70&agr; S6 kinase described (Pullen et al., 1997
FEBS Letters
410:78-82).
p70&agr; S6 kinase is an enzyme which lies downstream of phosphoinositide 3-kinases (P13-kinase). The mechanisms regulating the p70&agr; S6 kinase have not been fully elucidated. P13-kinase has recently been shown to activate another phosphoinositide-dependent protein kinase, termed PDK-1. So far, only PDK-1 has been shown to phosphorylate p70&agr; S6 kinase in vivo, and this phosphorylation is essential for p70&agr;
S6k
activity towards ribosomal S6 protein. Wortmannin, a fungal inhibitor which down-regulates the p70&agr; S6 kinase, is believed to act by inhibiting PI-3 kinase. In contrast, another fungal inhibitor, rapamycin, inhibits the p70&agr; S6 kinase by another cascade pathway involving the mammalian target of rapamycin (mTOR; also known as RAFT or FRAP) (Proud, 1996; Stewart et al., 1994
BioEssays
16:809-815). mTOR is a member of the PIK-related family of protein kinases (Pullen et al., 1997). Additional regulators of the p70&agr; S6 kinase include, but are not limited to protein kinase B (PKB), Cdc42, and Rac. The role of most of these proteins as p70&agr; S6 kinase regulators has yet to be fully elucidated.
SUMMARY OF THE INVENTION
The present invention is based on our discovery of a new gene which encodes a novel S6 kinase (p70&bgr;
S6k
). The invention includes isolated nucleic acid molecules selected from the group consisting of an isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID No.2, (e.g., SEQ ID No.1) an isolated nucleic acid molecule that encodes a fragment of SEQ ID No.2, an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule comprising SEQ ID No.1 under conditions of sufficient stringency to produce a clear signal and an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule that encodes the amino acid sequence of SEQ ID No.2 under conditions of sufficient stringency to produce a clear signal.
The present invention further includes the nucleic acid molecules operably linked to one or more expression control elements, including vectors comprising the isolated nucleic acid molecules. The invention further includes host cells transformed to contain the nucleic acid molecules of the invention and methods for producing a protein comprising the step of culturing a host cell transformed with the nucleic acid molecule of the invention under conditions in which the protein is expressed.
The invention further provides an isolated polypeptide selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ ID No.2, an isolated polypeptide comprising a fragment of SEQ ID No.2, an isolated polypeptide comprising conservative amino acid substitutions of SEQ ID No.2 and naturally occurring amino acid sequence variants of SEQ ID No.2.
The invention further provides an isolated antibody that binds to a polypeptide of the invention, including monoclonal and polyclonal antibodies and fragments thereof.
The invention further provides methods of identifying an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2 comprising the steps of: exposing cells which express the nucleic acid to the agent; and determining whether the agent modulates expression of said nucleic acid, thereby identifying an agent which modulates

Affiliated with

Also associated with

Rating

Identification and functional characterization of a novel... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Identification and functional characterization of a novel..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Identification and functional characterization of a novel... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3304816