Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-02-13
2002-11-05
Priebe, Scott D. (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C435S325000, C435S455000, C536S023100, C536S023500
Reexamination Certificate
active
06475778
ABSTRACT:
BACKGROUND OF THE INVENTION
Cellular interactions can be viewed as proceeding in two steps. Initially, an extracellular molecule binds to a specific receptor on a target cell, converting the dormant receptor to an active state. Subsequently, the receptor stimulates intracellular biochemical pathways leading to a cellular response, which may involve progression through the cell cycle, as well as changes in cellular gene expression, cytoskeletal architecture, protein trafficking, endocytosis, cell adhesion, migration, proliferation and differentiation, among others. An intracellular biochemical pathway which mediates some of these cellular responses involves members of the c-src family of protein tyrosine kinases, such as pp60
c-src
. Src tyrosine kinases transduce extracellular signals as diverse as responses to growth factors (for example, platelet derived growth factor (PDGF), epidermal growth factor (EGF)), antigens, cytokines, extracellular matrix molecules, among others. These extracellular signals give rise to a myriad of cellular responses, such as mitotic function, activation of Ras dependent pathways, phosphatidyl inositol 3-kinase activation and cytoskeletal reorganization.
The amino terminus of pp60
c-src
contains two motifs of approximately 100 and 60 amino acids in length named Src homology 2 and 3 domains (SH2, SH3), respectively. SH2 and SH3 domains have been identified in numerous signal transduction proteins (Pawson, T. and J. Schiessinger (1993)
J. Curr. Bio
. 3:434-442; Courtneidge et al. (1994)
Trends Cell Biol
. 4:345-347; Pawson, T. (1995)
Nature
373: 573-580). These domains presumably function as modular units that interact with other signal transduction proteins. The importance of SH2 and SH3 domains in signal transduction is underscored by the identification of “adapter proteins”, such as c-crk (Reichman et al., 1992), c-nck (Chou et al., 1992) and grb-2/ASH (Margolis et al., 1992; Matuokà et al., 1992), which lack a catalytic domain, and thus, appear to function as adaptors between membrane signaling and multiple downstream targets.
Proteins containing SH2 domains control biochemical pathways as diverse as phospholipid metabolism, tyrosine phosphorylation and dephosphorylation, activation of Ras-like GTPases, gene expression, protein trafficking and cytoskeletal architecture (Pawson, T. and J. Schlessinger (1993)
J. Curr. Bio
. 3:434-442). In vivo, SH2-containing proteins bind to phosphotyrosine (pTyr)-containing sites on activated receptors and cytoplasmic phosphoproteins (Anderson et al. (1990)
Science
250:979-982; Matsuda et al. (1990)
Science
248:1537-1539; Valius, M. and A. Kazlauskas (1993)
Cell
73:321-334). Indeed, crystal structures of the SH2 domains show a pocket configuration of amino acids that interact directly with a phosphotyrosine residue of an associated protein. Based on the crystal structure, the amino acid residues adjacent to the residues in direct contact with the phosphotyrosine determine the specificity of the interaction (Waksman et al. (1993)
J. Cell
72:779-790; Lee et al. (1994)
Structure
2:423-438).
SH3 domains have been found in a number of proteins involved in tyrosine kinase signaling, but also in cytoskeletal components and subunits of the neutrophil cytochrome oxidase, among others (Drubin et al. (1990)
Nature
343:288-290; Leto et al. (1990)
Science
248:727-730). In contrast to SH2 domains which interact with phosphorylated tyrosine residues of an associated protein, phosphorylation does not appear to be necessary for a protein to interact with a SH3 domain. The first SH3 binding protein identified, 3bp-1, shows homology to rho GTPase activating protein (GAP) (Cicchetti et al., (1992)
Science
257:803). C3G was initially identified as a GTP exchange factor for several G proteins, and was subsequently shown to have affinity for the SH3 domains of Crk and Grb-2 (Tanaka et al. (1994)
Proc. Natl. Acad. Sci. USA
91:3443-3447). G proteins themselves may be the targets for the binding of SH3 containing proteins. As an illustration, the proline rich C-terminus of the brain specific form of dynamin binds to several SH3 domains including those found in pp60
c-src
and pp59
c-fyn
, but not pp58
c-fgr
(Gout et al., 1993; Seedorf et al. (1994)
J. Biol. Chem
. 269:16009-16014). Dynamin is a microtubule-associated GTPase that is involved in endocytosis (Takel et al., 1995; Hinshaw et al., 1995). The binding of a SH3 domain to dynamin results in an increase in intrinsic GTPase activity (Gout et al., 1993).
SH3-binding sites consist of proline-rich peptides of approximately 10 amino acids (Ren et al. (1993)
Science
259:1157-1161; Yu et al. (1994)
Cell
76:933-945), which bind to isolated SH3 domains with dissociation constants of 5-100 &mgr;M (ref. 25). Recent structural and mutagenic analysis of peptide-SH3 complexes (Feng et al. (1994)
Science
266:1241-1246; Lim et al. (1994)
Nature
372:375-379; Musacchio et al. (1994)
Nature Struct. Biol
. 1:546-551; Wittekind et al. (1994)
Biochemistry
33:13531-13539; Rickles et al. (1994)
EMBO J
. 13:5598-5604) shows that peptides associated with SH3 domains adopt a left-handed polyproline type II helix, with three residues per turn, as illustrated by a PXXP consensus sequence (P=Proline, X=any amino acid) that forms a polyproline type II helix (Yu et al. (1994)
Cell
76:933-945). Solution and crystal structures of SH3 domains complexed with small peptides indicate a groove in the SH3 domain where the prolines of the PXXP helix are situated (Lim et al. (1994)
Nature
372:375-379; Yu et al. (1994)
Cell
76:933-945; Musacchio et al. (1994)
Nature Struct. Biol
. 1:546-551). Residues adjacent to the prolines also form contacts within the SH3 sequence and these interactions determine the specificity between a protein and a particular SH3 domain. For example, the arginine in “
R
PL
P
XX
P
” forms a salt bridge with aspartate at position 99 of pp60
c-src
. However the C-terminal arginine in the sequence “AFA
P
PL
P
R
R
” contacts the identical aspartate in pp60
c-src
, indicating that proteins may interact with SH3 domains in either a “plus” or “minus” orientation (named “class I” and “class II” binding, respectively; Yu et al. (1994)
Science
258:1665; Lim et al. (1994)
Nature
372:375-379).
Several proteins that interact with the SH3 domains of src-family kinases have been shown to be implicated in cellular growth. These include the regulatory subunit of phosphatidyl-inositol-3-kinase, p85 (Prasad et al. (1993)
Proc. Natl. Acad. Sci. USA
91:2834-2838), SHC (Weng et al., 1994), and ras GTPase-activating protein (Briggs et al., 1995). Furthermore, mutants within the SH3 domains of the adapter proteins c-crk and grb-2 inhibit v-abl oncogenic activity presumably by acting as “dominant negative” signal transduction effectors (Tanaka et al. (1995)
Proc. Natl. Acad Sci. USA
91:3443-3447).
Despite much progress in characterizing the signal trasnduction pathways involving SH3 domains, there is a great need for identifying novel mediators of these pathways, and in particular, binding proteins that interact with these SH3 domains. The identification of these novel molecules may provide for a detailed analysis of the amino acid contacts that determine the binding affinity and specificity of SH3 domains with an associated protein, which may in turn facilitate the development of therapeutic agents to be used in treating a diverse number of disorders.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of nucleic acid molecules which encode a novel family of src SH3 binding proteins, referred to herein as “differentiation enhancing factors” or “DEF polypeptides”. The DEF molecules show a highly conserved N-terminal domain and divergent C-terminus. The N-terminal domain preferably includes several structural motifs such as at least one src SH3 consensus binding sequence, at least one, and preferably four ankyrin repeats, at least one zinc finger domain, at least one pleckstrin homology domain and at least one C2 domain.
Chan Joanne
Harris David F.
Hu Erding
King Frederick J.
Roberts Thomas M.
Dana-Farber Cancer Institute
Kaushal Sumesh
Mandragouras Amy E.
Priebe Scott D.
Smith DeAnn F.
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