Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-06-29
2001-10-09
Priebe, Scott D. (Department: 1633)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S324000, C530S352000
Reexamination Certificate
active
06300473
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of signal transduction proteins, in particular, proteins related to Sam68.
BACKGROUND OF THE INVENTION
The binding of ligands to cell surface receptors can stimulate specific enzymatic activities in the cell. In certain cases, the ‘signal’ is transduced directly by the receptor which becomes capable of carrying out certain enzymatic functions. In other cases, the receptor triggers other proteins to carry out such enzymatic functions. The proteins which are modified by such activity are known as signal transduction proteins. Enzymatic activity stimulated includes tyrosine kinase (Pawson, Nature 373:573, 1995) and arginine methylase activity (Gary and Clarke, 1998, Prog Nucleic Acid Res. Mol. Biol. 61:65). It is known that many growth factor receptors and soluble tyrosine kinases are oncogenes and can transform cells. Thus, substrates of tyrosine kinases or the presence of post-translation modifications, such as arginine methylation, in response to growth factor receptors can serve as an indication of whether or not a cell is cancerous.
Signal transduction molecules often contain proline motifs and phosphotyrosine residues that serve as specific binding sites for Src-homology-3 (SH3) and Src-homology-2 (SH2) domain containing proteins (Pawson, Nature 373:573, 1995). SH2 recognizes peptides bearing phosphotyrosine (pTyr), and SH3 recognizes sequences containing one or more proline residues.
Sam68 (Src substrate activated during mitosis of 68 kDa), previously called p62 (GAP associated protein of 62 kDa) (Wong et al. (1992) Cell 69:551) is a signal transduction protein. It associates with SH2 and SH3 binding domains, for example those contained in certain tyrosine kinases. It can also bind single-stranded RNA (ssRNA), single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).
SUMMARY OF THE INVENTION
In one aspect, the invention provides a purified and isolated polypeptide comprising an amino acid sequence of at least 20 consecutive amino acids from the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
In another aspect, the invention provides a polynucleotide encoding a polypeptide of the invention.
In another aspect, the invention provides an expression vector comprising a polynucleotide of the invention.
In a further aspect, the invention provides a cell which has been transformed to express a polynucleotide of the invention.
In another aspect, the invention provides an antibody capable of specifically binding to a polypeptide of the invention.
In another aspect, the invention provides an animal cell which has been genetically modified such that its endogeneous gene coding for SLM-1 is incapable of expression. In another aspect, the invention provides an animal cell which has been genetically modified such that its endogeneous gene coding for SLM-2 is incapable of expression.
In a further aspect, the invention provides an animal which has been gentically modified such that its endogenous gene coding for SLM-1 is incapable of expression in certain tissue specific cells. In a further aspect, the invention provides an animal which has been gentically modified such that its endogenous gene coding for SLM-2 is incapable of expression in certain tissue specific cells.
In another aspect, the invention provides a commercial package for the assay of tyrosine kinase activity comprising a polypeptide of the invention together with instructions for its use.
In another aspect, the invention provides a commercial package for the assay of tyrosine kinase activity comprising an antibody of the invention, together with instructions for its use.
DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
SLM-1 and SLM-2 are Sam68-like mammalian proteins.
SLM-1 is a substrate for both tyrosine kinases and protein arginine methyltransferases and it has the property of binding many SH3 and SH2 domain containing proteins including Src kinases, Grb2, p120rasGAP and PLCg1. SLM-1 is a protein that is expressed ubiquitously in mammalian cells and functions as an RNA binding protein, binding poly U and poly A homopolymeric RNA.
SLM-2 is an RNA binding protein that shares the same signaling motifs as SLM-1 and Sam68. It is a substrate for both certain tyrosine kinases and protein arginine methyltransferases and it has the property of binding many SH3 and SH3 domain containing proteins. It binds poly G and poly A homopolymeric RNA. It is expressed mainly in the brain and skeletal muscle.
Peptide Sequences. The present invention provides for purified SLM-1 as well as SLM-1 derivatives that possess the biological and/or immunological properties of SLM-1. The present invention also provides for purified SLM-2 as well as SLM-2 derivatives that possess the biological and/or immunological properties of SLM-2.
The subject invention also encompasses polynucleotides that have the biological activity of SLM-1 or SLM-2 and an amino acid sequence identical or homologous to SLM-1 or SLM2, respectively. The nucleotide sequences encoding homologous proteins are capable of hybridizing to the nucleotide sequence encoding SLM-1 or SLM-2 under low stringency conditions, such as 40%-50% formamide, 37-42° C., 4X SSC, and wash conditions (after several room temperature washes with 2X SSC, 0.5% SDS) of stringency equivalent to 37° C. with 1×SSC, 0.05% SDS.
The biological activities of SLM-1 and SLM-2 include: (1) the property of serving as a substrate for one or more enzymes with tyrosine kinase activity, such as p60Src and p59fyn, (2) the property of serving as a substrate for enzymes with arginine methyltransferase activity, including such enzymes as protein arginine N-methyl transferases including PRMT1, PRMT2, and PRMT3, (3) the property of binding Src kinases, Src derivatives, or molecules with tyrosine kinase activity, e.g., when at least partially tyrosine phosphorylated, (4) the property of binding SH3 and SH2 domain containing proteins including Grb2, PLCg1, p120rasGAP and Grb2, e.g., when at least partially tyrosine phosphorylated, and (5) the property of associating with RNA.
SLM-1 and SLM-2 may be phosphorylated or non-phosphorylated as well as methylated or non-methylated. Phosphorylated SLM-1 or SLM-2 possess one or more phosphorylated tyrosine residues; methylated SLM-1 or SLM-2 possess one or more methylated arginine residues.
The amino acid sequences of mouse SLM-1 and SLM-2 were determined and are given herein as SEQ ID NO:1 and SEQ ID NO:2, respectively. It will be appreciated that SLM-1 and SLM-2 from species other than mouse, including humans, may have amino acid sequences that differ from the mouse sequences, but still possess SLM-1 or SLM-2 biological activity.
SLM-1 or SLM-2 derivatives include polypeptides possessing SLM-1 or SLM-2 biological activity and/or SLM-1 or SLM-2 immunological activity, respectively. A polypeptide possessing SLM-1 immunological activity can specifically bind with antibodies specific for SLM-1, or can, upon injection with suitable adjuvants, be used to induce an immune response specific for SLM-1. A polypeptide possessing SLM-2 immunological activity can specifically bind with antibodies specific for SLM-2, or can, upon injection with suitable adjuvants, be used to induce an immune response specific for SLM-2.
Derivatives of SLM-1 or SLM-2 include polypeptides which have the amino acid sequence of SLM-1 or SLM-2, but with one or more amino acid substitutions. It will of course be understood, without the intention of being limited thereby, that a variety of substitutions of amino acids is possible while preserving the structure responsible for the biological activity of the proteins disclosed herein. It is thus expected, for example, that interchange among non-polar aliphatic neutral amino acids, glycine, alanine, proline, valine and isoleucine, would be possible. Likewise, substitutions among the polar aliphatic neutral amino acids, serine, threonine, methionine, asparagine and glutamine could possibly be made. Substitutions among the charged acidic amino acids, aspartic acid and glutamic acid, could probably be made
BakerBotts LLP
Priebe Scott D.
Schnizer Richard
The Sir Mortimer B. Davis - Jewish General Hospital
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