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It has been unequivocally established that the receptor for peptides of the bombesin family is coupled to a G-protein(s) and does not possess intrinsic tyrosine kinase activity. Recently, however, bombesin has been shown to rapidly increase tyrosine phosphorylation of multiple substrates the most predominant of which migrate with an apparent Mr of 110,000-130,000 and 70,000-80,000 in intact quiescent Swiss 3T3 cells (Zachary et al., 1991a). Vasopressin, endothelin and bradykinin elicit a similar response (Leeb-Lundberg and Song, 1991; Zachary et al., 1991a). Furthermore, the stim ulation of tyrosine phosphorylation by neuropeptides is due to activation of cellular tyrosine kinase(s) (Zachary et al,, 1991b). The substrates for neuropeptide tyrosine phosphorylation in these ceils appear to be unrelated to known targets for the PDGF

receptor including the GTPase-activating protein (GAP), the PIP2-PLC-Y and

PI3K (Zachary et ai., 1991a). The identity of tyrosine phosphorylated proteins stimulated by bombesin and their coupling to the activated receptor is clearly of interest and is discussed below.

1 .5.8 .a Focal adhesion kinase (p 125^ ^ *^ )

The mechanism by which the src family of retroviral oncogenes causes malignant transformation has long proved elusive mainly because of the difficulty

in identifying substrates for the v-src p60 PTK. One approach has been the

generation of monoclonal antibodies to individual proteins which are tyrosine phosphorylated in chicken embryo fibroblasts (CEFs) transformed by Rous

Sarcoma Virus or expressing activated variants of src (Kanner et al., 1990). In

this way, several potential substrates were identified and the antibodies subsequently used to molecularly clone the corresponding complementary DMAs from expression libraries. This approach yielded the unexpected result that one such substrate, p i 25, is itself a novel type of PTK (Schaller et al., 1992a). This new kinase co-iocaiized with several components of cellular focal adhesions, such as tensin, vincuiin, and taiin which are important in regulating cytoskeletal structure and probably signal transduction (Schaller et al., 1992a). It was

therefore named focal adhesion kinase (p12sFAK) and was subsequently shown to be regulated by activation of the adhesive receptors of the integrin fam ily (Burridge et al., 1992; Guan and Shalloway, 1992; Kornberg et al., 1992; Lipfert

et al., 1992; Vuori and Ruoslahti, 1993) . It is now recognized that p1 2 5^A K ig a

m ajor substrate for not only bom besin-, vasopressin-, endothelin- and bradykinin-, but also LPA-, sphingosine- and PD G F-stim ulated tyrosine phosphorylation in Swiss 3T3 cells (Zachary et al., 1992; Leeb-Lundberg et al., 1994; Rankin and Rozengurt, 1994; Seufferlein and Rozengurt, 1994a;

Seufferlein and Rozengurt, 1994b). Thus, p12sFA K appears to be a point of

convergence in the action of neuropeptides, other growth factors, integrins and oncogenes and could be involved in the regulation of cell shape, adhesion and motility (reviewed in (Zachary and Rozengurt, 1992; Rozengurt, 1995)).

The cDNAs encoding p125^AK from 4 different species have been described

and the deduced amino acid sequences reveals extensive (approximately 91%) homology ((Schaller and Parsons, 1994)). The high degree of sequence

conservation amongst species and the fact that p1 2 5^A K jg expressed in most

tissues suggests that this novel PTK might be invoived in fundamentally important

signais. Structurally, p1 2 5^AK ggn be divided into 3 domains. The central catalytic

domain exhibits all conserved motifs charateristic of protein tyrosine kinase (PTK) domains. It is the fianking C- and N-terminal domains each approximately

400 amino acids long which make p1 2 5^A K gg different from most other PTKs.

Strikingly, they do not possess any motif determinant for either membrane association, such as acylation sites, or association with other proteins, such as the SH-2 or SH-3 domains. The C-terminal domain functions both as a focal adhesion targeting sequence or FAT (Hildebrand et al., 1993) and as a paxillin-binding site (Schaller and Parsons, 1994). The function of the N-terminal domain is not entirely clear but probably involves integrin receptor binding (Schaller and Parsons, 1994).

The precise regulation and function of p1 2 5^AK jg ggt clearly understood. It

is known that p1 2 5^A K rngy pg phosphorylated not only on a number of different

tyrosine residues but also on some serine residues (Schaller et al., 1993; Schlaepfer et al., 1994). It appears that the enzyme autophosphorylates

predominantly on tyrosine residue 397 both in vivo and in vitro (Schaller et al.,

1993). Phosphorylation of this site only slightly increases the ability of p1 2 5^A K

to tyrosine phosphorylate a synthetic substrate (Schaller and Parsons, 1994). Furthermore dephosphorylation with a protein tyrosine phosphatase has little

effect on p1 2 5^AK activity in vitro (Schaller and Parsons, 1994). These and other

data (Chan et al., 1994) suggest that tyrosine phosphoryiation of p1 2 5*^AK jg ggt

In contrast, tyrosine phosphorylation of residue 397 is important for

P I2 5FAK to be associated with the tyrosine phosphorylation of other proteins

which localise to focal adhesions such as paxillin and tensin in vivo (Schaller and

Parsons, 1994). A number of models could explain this, including the recruitment of a second PTK responsible for phosphorylating these cytoskeletal proteins. In

fact, tyrosine 397 has been shown to be a docking site for the SH-2 domain of v-

src p60 in src-transform ed CEFs and p12sF A K complexes with c-fyn p59 in

normal CEFs (Cobb et al., 1994). In normal cells, src p60 is regulated by the

interaction of its tyrosine-phosphorylated, negative-regulatory carboxy terminus with its own SH-2 domain. As peptides containing the sequence flanking tyrosine

397 within p1 2 5*^AK exhibit a higher affinity for the src p60 SH-2 domain than

do peptides containing the carboxy-terminal phosphorylated tyrosine residue of

src p60, it is tempting to speculate from these data that autophosphorylation of

P I2 5FAK rnay not only recruit src family members to focal adhesions but also

participate in the activation of src proteins. In this way p i2 5^ AK Q^d src p60

could act as a dual tyrosine kinase (Schaller and Parsons, 1994). As a consequence

of src p60 mediated phosphorylation of additional tyrosine residues on p1 2 5^AK

it could be envisaged that p1 25*^AK associates with other SH-2 containing proteins.

Interestingly, integrin activation in NIH3T3 cells results in both c -s rc

p60 and G rb-2 association with p1 2 5^ A K (S chlaepfer et al., 1994).

Furthermore, using p1 2 5^AK jmmunoprecipitates from src transformed NIH3T3

cells p1 2 5^AK appears to associate with Grb-2 and Sos. Indeed, Grb-2 binds to

tyrosine 925 of p1 2 5^AK jp vitro (Schlaepfer et al., 1994). Since integrins are

known to activate ras p21 and MAPK it is plausible that they may utilize p i 2 5*^AK

rather than a RTK to initiate the Grb2/Sos/Ras/Raf/MEK/MAPK pathway to the nucleus (Schlaepfer et al., 1994). Integrin activation also results in the binding and stimulation of PI3K in NIH3T3 cells and this interaction may be mediated via the SH-2 domain of the p85 subunit (Chen and Guan, 1994). The role of these

interactions in p1 2 5^A K function is not yet clear for integrin signalling and is as

yet undefined for neuropeptide signalling. However, it is now possible to see how p1 2 5^ A K may p© important in mediating many different signals including those directing cell proliferation.

1.5.8.b. The coupling of bombesin receptors to tyrosine