Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-06-04
2001-03-27
Borin, Michael (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S194000, C514S002600, C514S012200
Reexamination Certificate
active
06207393
ABSTRACT:
BACKGROUND OF THE INVENTION
(1) Field of the Invention
This invention relates to intracellular proteins involved in signal transduction and, more particularly, to peptides and methods for blocking signal transduction by binding to 14-3-3 proteins.
(2) Description of the Related Art
The family of proteins known as 14-3-3 proteins are expressed in a wide variety of organisms and tissues and at least seven different isoforms exist in mammalian cells (Aitken et al.,
Trends Biochem Sci
17:498-501, 1992; Aitken,
Trends Biochem Sci
20:95-97, 1995 which are incorporated by reference). The 14-3-3 proteins appear to mediate a number of biological activities. This protein has been found to activate neuronal tyrosine and tryptophan hydroxylase (Ichimura et al.,
Proc Natl Acad Sci USA
85: 7084-8, 1988 which is incorporated by reference); to regulate the activity of protein kinase C (Isobe et al.
FEBS Letters
308:121-124, 1991; Toker et al.
Eur J Biochem
191: 421-9, 1990; Tanji et al.,
J. Neurochem
63: 1908-16, 1994 which are incorporated by reference); as well as to bind to and presumably regulate a number of signaling proteins including Raf-1, polyoma middle T antigen, bcr, and PI-3 kinase (Fantl et al.,
Nature
371: 612-4, 1994 which is incorporated by reference).
One of these proteins, Raf, constitutes a family of serine/threonine kinases involved in the transduction of signals for growth and development from the cell surface to the nucleus. Members of the Raf family of kinases include Raf-1 which is ubiquitously expressed and A-Raf and B-Raf which have restricted patterns of expression. Raf kinases are believed to be key mediators of mitogenesis and differentiation, acting through a cascade of protein kinases that is also thought to be the pathway utilized by most oncogenes in cell transformation. (See Daum et al.,
Trends Biochem Sci
19:474-480, 1994 which is incorporated by reference.) The activation of Raf-1 in thought to involve phosphorylation of the molecule and several phosphorylation sites have been identified. (Morrison et al.,
J Biol Chem
268:17309-16, 1993 which is incorporated by reference). The phosphorylated Raf protein may then bind to the 14-3-3 protein which has been suggested to be essential to activation of Raf-1 in its mediation of these events inasmuch as microinjection of 14-3-3 results in Raf-1 activation and is required for function when Raf-1 is expressed in yeast (Fantl et al., supra; Li et al.,
EMBO J
14: 685-96, 1995; Irie et al.,
Science
265:1716-1719 which are incorporated by reference). It has been suggested that binding of 14-3-3 to Raf-1 is not necessary for activation, inasmuch as another group of investigators have reported that mutant forms of Raf-1 that are unable to bind to the 14-3-3 protein nevertheless show in vitro kinase activity as well as the ability to induce meiotic maturation in oocytes thus suggesting that binding to 14-3-3 is not essential (Michaud et al.,
Mol Cell Biol
15:3390-3397, 1995 which is incorporated by reference). This work is based upon the idea that 14-3-3 binds to a phosphorylation site (ser-259) that is induced by growth factor treatment. But 14-3-3 is associated with Raf-1 constitutively so this phosphorylation site (ser-259) cannot be the only binding site for 14-3-3. Thus, it was not appreciated that 14-3-3 binds to a Raf-1 phosphorylation site that is essential for function as is disclosed herein.
Nevertheless, because of the role of Raf-1 and possibly also the 14-3-3 proteins in a disease process involving growth and differentiation, in particular such conditions as cancer, atherosclerosis and autoimmune disease, it would be desirable to provide a method for interrupting this signal transduction pathway and thereby provide a new approach to treating these diseases.
BRIEF DESCRIPTION OF THE INVENTION
Accordingly, the inventors herein have succeeded in devising novel compositions and methods that act to inhibit the activation of a signal transducing protein by 14-3-3 protein by binding to the 14-3-3 protein and thereby prevent its binding to the signal transducing protein and its subsequent activation. Because the 14-3-3 proteins are utilized in a ubiquitous manner in the activation of signal transducing proteins, the present methods and compositions are applicable to a wide variety of such signal transducing proteins.
In one embodiment, the present invention provides a composition comprising an isolated phosphoserine-containing peptide comprising the amino acid sequence Arg-Ser-Xaa
1
-Xaa
2
-Xaa
3
-Pro where Xaa
1
is any amino acid, Xaa
2
is a phosphorylated serine and Xaa
3
is any amino acid (SEQ ID NO: 1). This sequence is based upon the amino acid residues surrounding both the Serine-259 (residues 256-261) and Serine-621 (residues of 618-623) of Raf-1. More particularly, peptides within the scope of this invention can contain the Serine-259 specific amino acid sequence Arg-Xaa
1
-Arg-Ser-Xaa
2
-Xaa
3
-Xaa
4
-Pro where Xaa
1
and Xaa
2
are any amino acid, Xaa
3
is a phosphorylated serine and Xaa
4
is any amino acid (SEQ ID NO: 2) or the Serine 621 specific amino acid sequence Lys-Xaa
1
-Xaa
2
-Arg-Ser-Xaa
3
-Xaa
4
-Xaa
5
-Pro where Xaa
1
, Xaa
2
and Xaa
3
are any amino acid, Xaa
4
is a phosphorylated serine and Xaa
5
is any amino acid (SEQ ID NO: 3). Peptides containing these sequences specifically bind to the 14-3-3 protein preventing the activation of a protein of the Raf-1 family of kinases that are signal transducing proteins. Other suitable peptides can be designed containing all or part of the 6 amino acids of SEQ ID NO: 1 so long as the Xaa
2
is a phosphorylated serine and the peptides are capable of binding to a 14-3-3 protein that activates a member of the Raf-1 family of signal transducing proteins. Similarly, such other suitable peptides which are capable of binding to a 14-3-3 protein that activates a Raf-1 family member can contain all or part of SEQ ID NO:2 or SEQ ID NO:3 so long as they contain a phosphorylated serine. Peptides containing 6 amino acids corresponding to SEQ ID NO: 1 in which the serine in position Xaa
2
is not phosphorylated show little or no capacity to bind to isoforms of the 14-3-3 protein.
Both peptide and non-peptide derivatives of the peptides can also be prepared that exhibit the functionality of being capable of binding to the 14-3-3 protein and blocking the binding of 14-3-3 to the signal transducing protein to inhibit its signal transducing properties in a cell.
In another embodiment, a method is provided for inhibiting the intracellular activation of a signal transducing protein by administering to a cell an effective amount of a peptide that contains the amino acids sequences as set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or a derivative thereof wherein the peptide or derivative thereof binds to a 14-3-3 protein. The administering of the exogenous peptide or derivative thereof to a cell and the binding of the peptide to the 14-3-3 protein blocks the binding of 14-3-3 to the signal protein resulting in inhibition of the signal transducing activity of the protein.
In another embodiment, a method is provided for identifying a substance that binds to a 14-3-3 protein and thereby inhibits the activity of a signal transducing protein. The method comprises forming a mixture comprising a construct containing a 14-3-3 protein or derivative thereof immobilized to a substrate, a labeled peptide containing a sequence obtained from a Raf-1 sequence surrounding serine-259 or serine-621, and said pharmacologic agent. The mixture is then incubated under conditions under which the labeled peptide can bind to the construct but for the presence of the pharmacologic agent. The binding of the labeled peptide is then determined wherein a decrease in binding of the labeled peptide indicates a binding of the pharmacologic agent to the construct. Also included within the scope of the present invention is the pharmacologic agent identified by this method. The method can also be used to isolate the pharmacologic agent such as from a library of chemical
Muslin Anthony J.
Shaw Andrey S.
Borin Michael
Howell & Haferkamp LC
Washington University
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