Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1998-06-30
2004-11-30
Ungar, Susan (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Reexamination Certificate
active
06824971
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular biology and cellular biology of cytokines. More specifically, the present invention relates to a methods of inhibiting or enhancing the TGF-&bgr;-Smad signaling pathway.
2. Description of the Related Art
The TGF-&bgr; family of polypeptide growth factors regulate cell division, differentiation, motility, adhesion and death in virtually all metazoan tissues
39,44,46,51,53,56
. Members of this family include the TGF-&bgr;s, the activins, the bone morphogenetic proteins (BMPs) and other related factors. Signal transduction by these factors involves three classes of molecules: a family of membrane receptor serine/threonine kinases, a family of cytoplasmic proteins, the Smad family, that serve as substrates for these receptors, and nuclear DNA-binding factors that associate with Smads forming transcriptional complexes
43,52
. Signaling is initiated by binding of the growth factor to a specific pair of receptor kinases, an event that induces the phosphorylation and activation of one kinase, known as the “type I receptor”, by the other kinase or “type II receptor”
65
. The activated type I receptor phosphorylates a subset of Smads, known as “receptor-regulated Smads” (R-Smads), which then move into the nucleus
43,52
. On their way to the nucleus, R-Smads associate with the related protein Smad4
9
, a tumor suppressor gene product
1
. In the nucleus, this complex may associate with specific DNA-binding proteins that direct it to the regulatory region of target genes. The first identified Smad-associated DNA-binding factor was the forkhead family member Fast1, which mediates activation of Mix.2 in response to activin-type signals during Xenopus embryogenesis
36
. The integrity of this signaling network is essential for normal development and tissue homeostasis, and its disruption by mutation underlies several human inherited disorders and cancer
43,52
.
Because of the diversity of processes controlled by different TGF-&bgr; family members, there is an intense interest in elucidating the basis for the specificity of their signal transduction pathways. The TGF-&bgr; and activin type I receptors, which have nearly identical kinase domains
31,60
, interact with and phosphorylate Smad2 (or the closely related Smad3)
16,40,30,54,8
which then interacts with DNA-binding factors such as Fast1
34,33,49
. The BMP receptors interact with Smad1 (or the closely related Smads 5, 8 or, in Drosophila, Mad)
35,40,11,14,18,10
which do not recognize Fast1
36
. Although the TGF-&bgr; and BMP pathways are well segregated from each other, their receptors and R-Smads are structurally very similar. The specificity of the receptor and Smad interactions in each pathway may therefore be dictated by discrete structural elements.
The Smad4/DPC4 tumor suppressor
1
is inactivated in nearly one half of pancreatic carcinomas
2
and to a lesser extent in a other cancers
2-4
. Smad4/DPC4, and the related tumor suppressor Smad2, belong to the Smad family of proteins which mediate TGF&bgr;/activin/bone morphogenetic protein (BMP)-2/4 cytokine superfamily signaling from the receptor serine/threonine protein kinases at the cell surface to the nucleus
5-7
. Smad proteins, which get phosphorylated by the activated receptor, propagate the signal, in part, through homo-oligomeric and hetero-oligomeric interactions
8,3
. Smad4/DPC4 plays a central role as it is the shared hetero-oligomerization partner of the other Smads. The conserved C-terminal domains of Smads are sufficient for inducing most of the ligand-specific effects, and are the primary targets of tumorigenic inactivation.
The conserved C-terminal domain of Smad family members is the likely effector domain, whereas the conserved N-terminal domain is the likely negative regulator of activity
14
. When overexpressed in a Smad4/DPC4−/− cell line, the Smad4/DPC4 C-terminal domain activates the transcription of TGF-&bgr; responsive genes and results in growth arrest in a ligand-independent manner, paralleling the effects of the TGF-&bgr; ligand
9
. In addition, microinjection of mRNAs encoding the C-terminal domain of Smad2 into Xenopus embryos can induce a mesoderm response that mimics the effects of the full-length protein
16
. Furthermore, the Smad4/DPC4-C-terminal domain fused to a heterologous DNA-binding domain can activate gene expression from a reporter construct
14
. Consistent with the Smad C-terminal domain being the main effector domain, the majority (10 out of 13) of the tumorigenic missense mutations in Smad4/DPC4 and Smad2, as well as mutations isolated from Drosophila and
C. elegans
genetic screens map to the C-terminal domain.
The prior art is deficient in the lack of effective means of inhibiting or enhancing the TGF-&bgr;-Smad signaling pathway. The present invention fulfills this longstanding need and desire in the art.
SUMMARY OF THE INVENTION
It is an object of the present invention to use the L3 loop of the Smad proteins 1, 2, 3, 4, 5 or 6 or the C-terminal tail of Smad proteins 1, 2, 3, 4 or 5 in protein-interaction assays to screen for agents that increase or decrease Smad interactions via these regions.
It is another object of the present invention to provide a method of screening for drugs that interfere with or enhance signaling by TGF-&bgr; or other members of the TGF-&bgr; family that signal through Smad proteins.
It is another object of the present invention to provide a screening method that utilizes high specificity peptide-Smad interactions and peptide receptor interactions and is suitable for adaptation to high throughput assays.
In one embodiment of the present invention, there is provided a method of screening for drugs which enhance or inhibit Smad binding to a complementary Smad via the L3 loop region, comprising the steps of: a) producing a synthetic Smad polypeptide encompassing the L3 loop region; b) attaching a detectable label onto this polypeptide; c) contacting the synthetic L3 loop polypeptide with a complementary Smad protein immobilized on a solid support; d) measuring the amount of labeled L3 loop polypeptide bound; e) in parallel to steps (c) and (d), conducting these same steps in the presence of a test substance; and f) comparing the amount of L3 loop polypeptide bound in the presence of a test substance with the amount bound in the absence of test substance so as to identify test substances that either increase L3 loop polypeptide binding to the Smad protein or decrease L3 loop polypeptide binding to the Smad protein.
In another embodiment of the present invention, there is provided a method of screening for drugs which enhance or inhibit Smad binding to a complementary Smad via the L3 loop region, comprising the steps of: a) producing a synthetic Smad polypeptide, encompassing the L3 loop region as defined by the crystal structure of the Smad4/DPC4 C-terminal domain; b) producing this polypeptide containing a chemical group that allows immobilization; c) contacting this L3 loop polypeptide with a labeled complementary Smad protein; d) measuring the amount of labeled Smad protein bound to the L3 loop polypeptide; e) in parallel to steps (c) and (d), conducting these same steps in the presence of a test substance; and f) comparing the amount of Smad protein bound in the presence of a test substance with the amount bound in the absence of test substance in order to identify test substances that either increase L3 loop polypeptide binding to the Smad protein or decrease L3 loop polypeptide binding to the Smad protein.
In yet another embodiment of the present invention, there is provided a method of screening for drugs which enhance or inhibit Smad4 binding to a complementary Smad via the C-terminal phosphorylated tail (“C-tail”) of this Smad, comprising the steps of: a) producing a synthetic polypeptide corresponding to the C-terminal tail of a given Smad encompassing the C-terminal tail that follows the H5 alpha-helix as defined by the crystal structure of the Smad4/DPC-terminal domain;
Massague Joan
Pavletich Nikola
Shi Yigong
Adler Benjamin Aaron
Sloan-Kettering Institute for Cancer Research
Ungar Susan
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