Shc-binding protein

Details Shc-binding protein Shc-binding protein

1999-07-29

2002-06-04

Carlson, Karen Cochrane (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

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

Reexamination Certificate

active

06399747

ABSTRACT:

FIELD OF INVENTION
The present invention relates generally to the identification and isolation of a novel Shc-binding proteins, to novel nucleic acid molecules encoding such polypeptides and more particularly to the isolation and identification of a unique Shc binding protein designated PAL
P
rotein expressed in
A
ctivated
L
ymphocytes), and nucleic acid molecules encoding PAL.
BACKGROUND
The ubiquitously expressed Shc adapter proteins play a role in coupling growth factor receptor activation to intracellular signaling pathways. The mammalian Shc gene encodes at least three overlapping proteins with molecular weights of approximately 46 kDa, 52 kDa and 66 kDa (also called the p46, p52, and p66 isoforms or proteins, Bonfini et al.,
TIBS
21:257-261 (1996); Migliaccio et al.,
EMBO J.
16:706-716 (1997); Pelicci et al.,
Cell
70:93-104 (1992)). All three protein products share a carboxy terminal Src Homology 2 (SH2) domain, a central glycine/proline rich domain with homology to alphal collagen (CH1), and an amino terminal phosphotyrosine binding (PTB) domain which is different from the SH2 domain. The p52 and p46 isoforms differ only by 46 amino acids at the extreme amino terminus and are generated by the use of alternative translation initiation sites (Pelicci et al.,
Cell
70:93-104 (1992)). The p66 isoform is produced via alternative splicing of the Shc gene and contains an amino terminal extension which encodes a second collagen homology region (CH2) in addition to the common PTB, CH
1
, and SH2 domains. Interestingly, it has recently been demonstrated that the expression of p66 is more restricted, and that some of its biological, properties are distinct from those of the p52 and p46 Shc isoforms (Bonfini et al.,
TIBS
21:257-261 (1996); Migliaccio et al.,
EMBO J.
16:706-716 (1997)).
Shc proteins are typically tyrosine phosphorylated following activation of receptor tyrosine kinases (van der Geer et al.,
Ann. Rev. Cell Biol.
10:251-337 (1994)), such as the epidermal growth factor receptor (EGFR) (Pelicci et al.,
Cell
70:93-104 (1992)), the platelet-derived growth factor receptor (PDGFR) (Yokote et al.,
J. Biol. Chem.
269:15337-15343 (1994)), the nerve growth factor receptor (TrkA) (Obermeier et al.,
EMBO J.
13:1585-1590 (1994)), the insulin receptor (Pronk et al.,
J. Biol. Chem.
268:5748-5753 (1993)), and erbB-2 (Segatta et al.,
Oncogene
9:2105-2112 (1993)), as well as following activation of receptors that lack intrinsic tyrosine kinase activity, such as the T-cell receptor (TCR) (Ravichandran et al., Science 262:902-905 (1993)), the B-cell receptor (Saxton et al.,
J. Immunol.
153:623-636 (1994)), the receptors for the interleukins (Burns et al.,
J. Biol Chem.
268:17659-17661 (1993); Cutler et al.,
J. Biol. Chem.
268:21463-21465 (1993); Ravichandran et al.,
J. Biol. Chem.
269:1599-1602 (1994)), and the erythropoietin receptor (Damen et al.,
Blood
82:2296-2303 (1993)). Additionally, tyrosine phosphorylation of Shc proteins has been detected after activation of G-protein coupled receptors (Cazaubon et al.,
J. Biol. Chem.
269:24805-24809 (1994); Chen et al.,
EMBO J.
15:1037-1044 (1996);al., Ptazniket et al.
J. Biol. Chem.
270:19969-19973 (1995); Touhara et al.,
Proc. Natl. Acad. Sci. USA.
92:9284-9287 (1995); van Biesen et al.,
Nature
376:781-784 (1995)), ligation of integrins (Maniero et al.,
EMBO J.
14:4470-4481(1995); Wary et al.,
Cell
87:733-743 (1996)), and in cells expressing activated Src, Fps, Sea or Lck (Baldari et al.,
Oncogene
16:1141-1147 (1995); Crowe et al.,
Oncogene
9:537-544 (1994); McGlade et al.,
Proc. Natl. Acad. Sci. USA
89:8869-8873 (1992); Pelicci et al.,
Oncogene
11:899-907 (1995)), which implicates Shc proteins as important substrates of cytoplasmic tyrosine kinases.
Shc Protein Binding
Shc proteins are able to directly bind to tyrosine-phosphorylated peptides or proteins, including activated receptor tyrosine kinases, typically by virtue of their SH2 or PTB domains (Bonfini et al.,
TIBS
21:257-261 (1996); Pawson,
Nature
373:573-579 (1995)). The Shc SH2 domain preferentially binds to tyrosine phosphorylated peptides in the sequence context pY-E/I -X-I/L/M (where X represents any amino acid) (Songyang et al.,
Mol. Cell. Biol.
14:2777-2785 (1994)) and mediates the binding of Shc to the PDGFR (Songyang et al.,
Mol. Cell. Biol.
14:2777-2785 (1994)), EGFR (Pelicci et al., Cell 70:93-104 (1992)), receptor tyrosine kinase (RET) (Pronk et al.
J. Biol. Chem.
268:5748-5753 (1993)), and the CD3 zeta chain (Ravichandran et al.,
Science
262:902-905 (1993)). The PTB domain of Shc proteins also recognizes tyrosine phosphorylated peptides, but in a different context by selecting specific residues amino terminal to the phosphorylated tyrosine (Songyang et al.,
J. Biol. Chem.
270:14863-14866 (1995)). The Shc PTB domain has been shown to bind directly to N-P-X- pY sequence motifs in the cytoplasmic domains of the EGFR (Blaikie et al.,
J. Biol. Chem.
269:23031-32034 (1994)), TrkA(Diklic et al.,
J. Biol. Chem.
270:15125-15129(1995)), RET (Lorenzo et al.,
Oncogene
14:763-771(1997)), and the IL-2R&bgr; chain (Ravichandran et al.,
Proc. Natl. Acad Sci. USA
28:5275-5280 (1996)).
Phosphorylated Shc proteins are also able to associate with the Grb2 adapter protein by binding of the Grb2 SH2 domain to the phosphorylated tyrosine residue 317 (Y317) within the CH1 domain of Shc proteins (Rozakis-Adcock et al.,
Nature
360:689-692 (1992); Salcini et al.,
Oncogene
9:2827-2836 (1994)). Grb2 is stably associated with the Ras guanine nucleotide exchange factor, SOS (Batzer et al.,
Nature
363:85-88 (1993); Buday et al.,
Cell
73:611-620 (1993); Chardin et al.,
Science
260:1338-1343 (1993); Egan et al.,
Nature
363:45-51 (1993); Rozakis-Adcock et al.,
Nature
363:83-85 (1993)), and membrane localization of the Grb2-SOS complex results in activation of Ras (Aronheim et al.,
Cell
78:949-961 (1994)). Therefore, it has been proposed that Shc proteins are involved in coupling cell surface receptors to Ras activation. Several studies on the effects of Shc protein over expression provide support for this hypothesis. First, co-expression of a dominant negative mutant of Ras blocks neurite outgrowth in PC12 cells induced by Shc protein over expression (Rozakis-Adcock et al.,
Nature
360:689-692 (1992)). Second, over-expression of Shc protein in NIH 3T3 fibroblasts results in transformation (Pelicci et al.,
Cell
70:93-104 (1992)), and this can be abrogated by mutation of the presumed Grb2 binding site (Salcini et a.,
Oncogene
9:2827-2836 (1994)). Also, over-expression of Shc protein enhances EGF induced activation of MAP kinases (Migliaccio et al.,
EMBO J.
16:706-716 (1997)), and cell motility and growth in response to hepatocyte growth factor (HGF) (Pelicci et al.,
Oncogene
10:1631-1638 (1995)).
Shc Protein Ras-independent Binding
The modular structure of Shc proteins permits their interaction with multiple signaling molecules, suggesting that Shc proteins could function to couple activated receptors to pathways other than Ras. Two additional sites of Shc protein tyrosine phosphorylation have recently been mapped to tyrosine residues 239 and 240 (Y239/240) (Gotoh et al.,
EMBO J.
15:6197-6204 (1996); Harmer et al.,
Mol. Cell. Biol.
17:4087-4095 (1997); van der Geer et al.
Curr. Biol.
6:1435-1444 (1996)). Tyrosine 239 is present within a Grb-2 SH2 binding motif, and has been demonstrated to associate with Grb-2 in vivo (Gotoh et al.,
Mol. Cell. Biol.
17:1824-1831 (1997); Harmer et al.
Mol. Cell. Biol.
17:4087-4095 (1997)). The Y239/240 phosphorylation sites may also couple Shc proteins to additional downstream SH2 containing proteins, since phosphopeptides corresponding to Y239/240 have been demonstrated to bind to a variety of as yet, unidentified proteins, in addition to Grb2 (van der Geer et al.,
Curr. Biol.
6:1435-1444 (1996)). A novel role for Shc proteins has been suggested in which phosphorylation of Y239/Y240 leads to c-myc induction, and suppression of apoptosis in Ba/F3 cells, in a R

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