Antibodies to a novel src-family kinase

Details Antibodies to a novel src-family kinase Antibodies to a novel src-family kinase

1999-01-12

2001-09-18

Saunders, David (Department: 1644)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

Blood proteins or globulins, e.g., proteoglycans, platelet...

C530S387900, C530S388260, C530S389100, C435S331000, C435S338000

Reexamination Certificate

active

06291651

ABSTRACT:

FIELD OF THE INVENTION
The present invention pertains to a novel src-family kinase, its role in embryonic development and in carcinogenesis. Amino acid and nucleic acid sequences of the novel src family kinase are disclosed. Methods of using the novel src family kinase are also included.
BACKGROUND OF THE INVENTION
The src family of non-receptor tyrosine kinases are a well-studied class of signaling molecules which heretofore have not been shown to play any role in mesoderm induction. All src-family proteins contain catalytic, tyrosine kinase domains, as well as src-homology 2 and src-homology 3 (SH2 and SH3) domains. These domains were first discovered in cytoplasmic (non-receptor) protein tyrosine kinases such as the src oncogene product, thus leading to the term ‘src homology domains’ [Sadowski et al.,
Mol Cell. Biol
6:4396-4408 (1986)]. src is the prototypical member of this gene family, and was the first characterized proto-oncogene: elimination of a ncegative regulatory tyrosine at the carboxy-terminus of src converts the molecule to a potent transforming agent, and all members of the family share this residue [Brown and Cooper,
Biochimica el Biophysica Aclt,
1287:121-149 (1996)]. Nine src-related genes have been cloned thus far, and several of these have been shown to be required for normal function of the immune and nervous systems [Brown and Cooper,
Biochimica et Biophysica Acta,
1287:121-149 (1996)]. No definitive role, however, has been described for these proteins during early development. SH2 and SH3 domains are two individual protein modules that play an intermediary role in eukaryotic cellular signal transduction. After the initiation of the signal by the binding of an extracellular ligand to a transmembrane receptor having an associated tyrosine kinase, SH2 and SH3 domains mediate many of the protein-protein interactions that are necessary for transmission of the signal [Cantlcy el al.,
Cell,
64:281-302 (1991); Schlessinger el al.,
Neuron,
9:383-391 (1992); Pawson el al.,
Curr. Biol.,
3:434-442 (1993)].
The unique importance of these domains became clear with the discovery of the crk oncogene product, which consists of little more than an SH2 and an SH3 domain fused to the viral gag protein, but is capable of transforming cells [Mayer el al.,
Nature,
332:272-275 (1988)]. SH2 and SH3 domains have been identified in molecules with distinct functions that act downstream from the receptors for, among others, epidermal growth actor (EGF), platelet-derived growth factor (PDGF), insulin and interferon, and the T-cell receptor [Koch el al.,
Science,
252:668-674 (1991)].
The key aspect of the function of SH2 and SH3 domains is their ability to recognize particular amino acid sequences in their target proteins: SH2 domains bind tightly to phosphorylated tyrosine residues [Andeirson el al.,
Science;
250:979-982 (1990); Matsuda el al.,
Science
248:15537-1539 (1990); Moran el al.,
Proc. Natl. Acad. Sci. USA
87:8622-8626 (1990); Mayer el al.,
Proc. Natl. Acad. Sci. USA,
88:627-631 (1991); Songyang et al.,
Cell
72:767-778 (1993)] whereas SH3 domains bind to unmodified peptide sequence that are rich in proline and hydrophobic amino acids [Cicchetti et al.,
Science
257:803-806 (1992); Ren el al.,
Science
259:1157-1161 (1993)]. The modular nature of these domains is made clear by the fact that they occur in different positions in the polypeptide chains of the intact proteins of which they are a part, and that the binding functions can often be reproduced by isolated domains. As indicated above, however, the role of src-family tyrosine kinases in the signalling pathway during embryonic development has been obscure. During embryogenesis, inductive interactions among cells underlie the development of much of the body plan. The process of mesoderm formation is a critical and well-characterized example of an early inductive event. In
Xenopus laevis,
factors secreted from the vegetal pole induce mesoderm in the adjacent marginal zone [Klein and
Melton, Endocr. Rev.,
15:326-341 (1994)]. During mesoderm induction, it is essential that information received by the marginal zone cells be communicated from the cell surface to the nucleus, where determination of cell fate is driven by an alteration in gene expression. Members of both the Transforming Growth Factor-b (TGF-b) and Fibroblast Growth Factor (FGF) ligand families appear to play essential roles in the formation of mesoderm [Klein and
Melton, Endocr. Rev,
15:326-341 (1994)]. The downstream effectors of these growth factors are distinct: TGF-b ligands signal through serine-threonine kinase receptors, whose effects are mediated through the recently characterized Smad proteins [Massague et al,
TICB,
7:187-192 (1997)]. The Smad pathway appears to be quite direct: although a number of positively and negatively acting Smads may interact in mesoderm induction, few other factors seem to be involved in the propagation of signal from cell surface to nucleus. Signaling through the FGF receptor tyrosine kinase, however, appears to be significantly more complex, involving a multiprotein interaction at the plasma membrane, and subsequent activation of the ras/MAP kinase pathway [Labonne and Whitman,
Dev. Biol.,
183:9-20 (1997), and references therein).
The importance of isolating and identifying the factors involved in early development cannot be emphasized. For example, screening for mutations of such factors, in utero, can serve as a powerful tool in early identification of developmental defects. Therefore, there is a need to isolate and identify factors that mediate the signaling initiated at the FGF receptor. Furthermore, there is need to obtain nucleic acid probes and antibodies which can be used to identify the absence of such factors and/or defects in such factors.
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
The present invention provides a unique src-family kinase (SFK) that plays a role in the transformation of uncommitted embryonic cells to mesodermal cells. Furthermore, this src-family kinase (also denoted as
laloo
) has properties that are consistent with it being a proto-oncogene.
One aspect of the invention provides an isolated nucleic acid encoding a src-family kinase comprising an amino acid sequence substantially homologous to that of SEQ ID NO:2, and having the following structural/functional domains: (i) a catalytic tyrosine kinase domain; (ii) a src-homology-2 (SH2) domain; and (iii) a src-homology-3 (SF13) domain. In a particular embodiment, the isolated nucleic acid encodes a src-family kinase that further comprises a tyrosine in the carboxyl-terminal portion of the protein which can act as a site of negative regulation. In a preferred embodiment the nucleic acid encodes a vertebrate SFK. In one particular embodiment of this type the nucleic acid encodes a human SFK, (
human laloo
).
In a more particular embodiment the isolated nucleic acid encodes a vertebrate src-family kinase (SFEK) comprising the amino acid sequence of SEQ ID NO:2. In another particular embodiment the isolated nucleic acid encodes a vertebrate SFK comprising the amino acid sequence of SEQ ID NO:2 with a conservative amino acid substitution. In a preferred embodiment of the present invention, the isolated nucleic acid encodes a
Xenopus laloo
and comprises the coding sequence of SEQ ID NO: 1.
In an alternative embodiment of this type the isolated nucleic acid contains a nonconservative amino acid substitution which alters a functional or regulatory property of the SFK. In one particular embodiment of this type, the tyrosine at position 492 in SEQ ID NO:2 is replaced with a pheniylalanine (Y492F). This tyrosine is a phosphorylatable site that is involved in the regulation of the SFK. In another such embodiment the arginine at position 259 in SFQ ID NO:2 is replaced with

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