Constitutively active transcription factors and their uses...

Details Constitutively active transcription factors and their uses... Constitutively active transcription factors and their uses...

1999-07-31

2001-05-22

Brusca, John S. (Department: 1631)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S350000, C536S023100, C435S320100, C435S068100

Reexamination Certificate

active

06235873

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to modified Stat proteins that in the absence of tyrosine phosphorylation are capable of constitutive dimerization, transcriptional activation, and induction in transfected cells of cellular transformation and tumorigenesis, as well as their utility in identifying agents capable of modulating cellular transformation and tumorigenesis.
BACKGROUND OF THE INVENTION
Stat (Signal Transducers and Activators of Transcription) proteins are latent transcription factors that become activated by phosphorylation on a single tyrosine (near residue 700), typically in response to extracellular ligands (Darnell, 1997; Stark et al., 1998; U.S. application Ser. Nos. 08/212,185, filed Mar. 11, 1994; Ser. No. 09/087,465, filed May 29, 1998; Ser. No. 08/951,130, filed Oct. 15, 1997; Ser. No. 09/012,710, filed Jan. 23, 1998; Ser. No. 08/820,754, filed Mar. 19, 1997; and U.S. Pat. No. 5,716,622; all of the foregoing incorporated herein by reference in their entireties). More than 40 different polypeptide ligands cause Stat phosphorylation through either cytokine receptors plus associated Jak kinases or growth factors (e.g. EGF, PDGF, CSF-1) acting through intrinsic receptor tyrosine kinases. An active Stat dimer is formed via the reciprocal interactions between the SH2 domain of the monomer and the phosphorylated tyrosine of the other (Chen et al., 1998). The dimers accumulate in the nucleus, recognize specific DNA elements and activate transcription. The Stat proteins are subsequently inactivated by tyrosine dephosphorylation and return to the cytoplasm (Haspel et al., 1996).
Ligand dependent activation of the Stats is often associated with differentiation and/or growth regulation while constitutive activation of Stats (i.e. activation without known requirements of extracellular polypeptides) is often associated with growth dysregulation. For example, the development of the antiviral state or growth restraint secondary to either IFN-&agr; of IFN-&ggr; treatment requires transcriptionally competent Stat1 (Bromberg et al., 1996; Horvath and Darnell, 1996) and mice lacking Stat1 form chemically induced tumors of the skin more easily than wild-type animals (Kaplan et el., 1998). Various stages of lymphocyte or monocyte development depend on Stats 3, 4 and 6 (Kaplan et al., 1996; Takeda et al., 1996; Thierfelder et al., 1996; Takeda et al., 1998; Takeda et al., 1999); development of breast epithelium requires Stat5A (Liu et al., 1997; Teglund et al., 1998), and proper male growth hormone response demands Stat5B (Udy et al, 1997; Teglund et al., 1998). A growing number of tumor-derived cell lines as well as samples from human cancers are reported to contain constitutively activated Stat proteins, very frequently Stat3 (Garcia and Jove, 1998). For example, all
src
transformed cell lines exhibit constitutively activated Stat3 (Yu et al, 1995). Moreover, dominant negative Stat3 suppresses
src
transformation without having any effect on
ras
transformation (Bromberg et al., 1998; Turkson et al., 1998). Cell lines from multiple myelomas that have become growth factor independent require constitutively active Stat3 to protect against apoptosis (Catlett-Falcone et al., 1999). A high proportion of head and neck cancers (often of squamous cell origin) have constitutively active Stat3, most likely secondary to aberrant EGF receptor signaling (Grandis et al., 1998) and dominant negative Stat3 slows the growth of cell lines developed from these cancers.
Oncogenes were first defined as viral or mutated cellular genes that could confer a transformed phenotype to cultured cells (Lodish et al., 1995). When oncogenes were characterized at the molecular level, many were found to be constitutively activated. One constitutively activated Stat molecule has been described. Amino acid changes in two separate domains of Stat5 results in constitutive activation and obviates the need for IL-3 in the growth of BaF3 cells (Onishi et al., 1998). However, the amino acids that are changed in the Stat5 molecule (H-R
299
and S-F
711
) are not conserved between Stat5 and Stat3.
It is toward the development of a dimerizable and constitutively active Stat protein particularly in the absence of tyrosine phosphorylation and its utility in investigating the role of the active protein in transcription and inducing cell transformation, and the effects of modulators thereon, that the present invention is directed.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.
SUMMARY OF THE INVENTION
In its broadest aspect, the present invention is directed to modified Stat proteins capable of constitutively dimerizing and binding to DNA in the absence of tyrosine phosphorylation. The modified Stat proteins have at least one cysteine residue provided in the Stat protein which is capable of interacting with the same cysteine residue on a second modified Stat protein, forming a dimer. Non-limiting examples of Stat proteins that may be so modified include Stat1 (SEQ ID NO:1), Stat2 (SEQ ID NO:2), Stat3 (SEQ ID NO:3), Stat4 (SEQ ID NO:4), Stat5 and Stat6. The first cysteine may be provided in a conserved domain of the Stat protein; for example, the C-terminal loop of the SH2 domain. Suitable positions include but are not limited to position 656 or 658 Stat1; position 706 or 707 of Stat1; position 653 or 655 of Stat2; position 725 or 726 of Stat 2; position 710 or 711 of Stat 3; position 662 or 664 of Stat3; position 651 or 653 of Stat4; position 699 or 700 of Stat 4; position 715 or 716 of Stat5; or position 697 or 698 of Stat6. In a further aspect of the invention, a second cysteine is provided in the modified Stat protein, for example, both aforementioned positions in the Stat molecules, i.e., position 656 and 658 of Stat1; position 706 and 707 of Stat1; position 653 and 655 of Stat2; position 725 and 726 of Stat 2; position 710 and 711 of Stat 3; position 662 and 664 of Stat3; position 651 and 653 of Stat4; position 699 and 700 of Stat 4; position 715 and 716 of Stat5; or position 697 and 698 of Stat6. In a preferred embodiment of the invention, the modified Stat protein is Stat3, wherein position 662 is C, and position 664 is C (SEQ ID NO:5). In a still further aspect, the modified Stat protein may have a third or a third and fourth cysteine residue.
In a further aspect of the invention, the modified Stat protein further comprises an epitope tag, for example, FLAG.
The invention is also directed to the polynucleotide sequences encoding the aforementioned modified Stat proteins, including those with an epitope tag.
In another broad aspect of the present invention, a method is provided for preparing a modified Stat protein capable of constitutively dimerizing and binding to DNA in the absence of phosphorylation. The method involves introducing in a Stat protein at least one cysteine; for example, by site-directed mutagenesis, wherein the cysteine of a first modified Stat protein molecule is capable of interacting with the same residue of a second modified Stat protein, forming a dimer. Non-limiting examples of suitable Stat proteins include Stat1 (SEQ ID NO:1), Stat2 (SEQ ID NO:2), Stat3 (SEQ ID NO:3), and Stat4 (SEQ ID NO:4). The first cysteine residue may be introduced into a conserved domain of said Stat protein, for example, the C-terminal loop of the SH2 domain. By way of non-limiting example, a cysteine residue introduced into the modified Stat protein is capable of interacting with and promoting dimer formation with the introduced cysteine residue on a second modified Stat protein. Examples of suitable positions include but are not limited to position 656 or 658 of Stat1; position 706 or 707 Stat1; position 653 or 655 of Stat2; 725 or 726 of Stat 2; position 710 or 711 of Stat 3; position 662 or 664 of Stat3; position 651 or 653 of Stat4; 699 or 700 of Stat 4; position 715 or 716 of Stat5; or position 697 or 698 of Stat6.
Any technique for mutagenesis known in the art can be used for provi

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