CONCLUSIONES Y RECOMENDACIONES

Recently, Lev and co-workers have identified an unanticipated role for N irl- 3 in Pyk2 signalling pathways (Lev et al., 1999). As indicated previously, N irl-3 bind to the amino-terminal domain of the protein tyrosine kinase Pyk2. Using both a yeast two hybrid approach and direct binding experim ents. Lev and co-workers found that the conserved carboxy-term inal domain of N irl-3 associated with the amino-terminal domain of Pyk2.

An association of N irl-3 with Pyk2 in vivo was confirmed by co-transfection studies in which haemagglutanin (HA)-tagged N irl-3 cDNA and Pyk2 were co­ transfected in to HEK293 cells (Lev et al., 1999). Furtherm ore N irl-3 could be detected in anti-Pyk2 immunoprecipitates prepared from the transfected cell lysates. Sim ilar studies, in which Pyk2 was replaced with focal adhesion kinase (FAK), demonstrated the specificity of the association between N irl, 2 or 3 and Pyk2 (Lev et

al., 1999). Additionally, Pyk2-Nir complexes were detected in lysates prepared from brain tissue.

Analysis of Pyk2 lysates from cells co-expressing Nir2 dem onstrated that both Nir2 and Pyk2 were tyrosine phosphorylated and are present in the same immunocomplex. However, while, Nir2 was not tyrosine phosphorylated in cells expressing a kinase-negative Pyk2 m utant protein (PKM), it was still found to associate with PKM (Lev et al., 1999). These data indicate that N irl-3 are likely substrates of Pyk2 and that tyrosine kinase activity is not required for association of these proteins. Endogenous Nir2 and Pyk2 were also found to co-immunoprecipitate from quiescent HL60 cell lysates following stimulation of the intact cells with agents know n to activate Pyk2, such as PM A and the ER Ca^^-ATPase inhibitor, thapsigargin (Lev et al., 1999). Furtherm ore, these agonists induced a strong phosphorylation of Nir2 tyrosine residues. W hile these data strongly suggest that endogenous Nir2 is a substrate for Pyk2, it is possible that tyrosine phosphorylation of Nir proteins is induced by another protein tyrosine kinase that can be activated by Pyk2, such as Src. These observations, together with expression of N irl-3 with Pyk2 in distinct regions of the rat brain and retina has led to the hypothesis that Pyk2 is an upstream regulator of Nir proteins (Lev et al., 1999).

1.9.5.a Pyk2

Protein tyrosine kinase (PTK) activities transduce many extracellular signals that trigger key cellular events (Avraham et al., 2000 and references therein), such as mitogenesis and cytoskeletal rearrangement, and thereby co-ordinate physiological processes, such as development and oncogenesis. PTKs mediate these responses by activating a variety of intracellular signalling pathways through their intrinsic kinase activity. Protein tyrosine kinases can be divided into receptor (RTKs) and non­ receptor kinases, based on the presence of extracellular ligand-binding and transmembrane domains in the former.

Pyk2 (also known as RAFTK, FAK2, CAK-B or CADTK) belongs to a new family of PTKs, which has been named after FAK (reviewed by Avraham et al.,

2000; Hanks and Poke, 1997). FAK and Pyk2 have m olecular weights of 110- 125kDa and exhibit around 48% amino acid identity. Both kinases have a similar domain structure: a unique amino-terminal domain, a central protein tyrosine kinase domain, and two proline rich regions at the carboxy-terminal (Avraham et al., 2000; Hanks and Poke, 1997). Despite their structural similarity, Pyk2 and FAK have

different tissue expressions and different modes of activation. Pyk2 is expressed mainly in the central nervous system and in cells derived from haem atopoietic lineages while FAK is expressed in a variety of tissues (Schlaepfer and Hunter, 1998). Immunolocalisation studies have demonstrated that FAK is localised to focal adhesion sites, while Pyk2 is mainly distributed throughout the cytoplasm and is concentrated in the perinuclear region (Hanks and Polte, 1997). Activation of FAK is linked with transmembrane integrin receptors and functions during integrin-mediated signalling pathways (reviewed by Schlaepfer and Hunter, 1998). In contrast, Pyk2 is activated by a variety of extracellular stimuli including growth factors, GPCR agonists, extracellular matrix proteins and stress signals, as well as stimuli, which elevate the intracellular Ca^^ concentration. The cell-dependent roles of Pyk2 in various signalling cascades have been recently reviewed (Avraham et a l , 2000) and will therefore not be discussed in detail here.

A large body of data indicates that Pyk2 regulates a variety of cellular responses, including neuronal excitability, T- and B-cell receptor signalling, cell growth and survival. Pyk2 is activated in response to stress signals, such as tumour necrosis factor-a, hyperosm otic shock and UV light, thereby inducing Jun N- terminal kinase (INK) activation (Tokiwa et al., 1996; Yu et al., 1996). In PC12 cells, Pyk2 tyrosine phosphorylation and activation are stim ulated by neuronal stimuli and stress signals leading to m odulation of the delayed-rectifier-type potassium channel and the IN K signalling pathway, respectively (Lev et a l, 1995; Yu et a l , 1996). Similar to FAK, Pyk2 is tyrosine phosphorylated and activated by adhesion-mediated signalling in platelets and B-cells (Astier et al., 1997; Tokiwa et al., 1996). Schlessinger and co-workers have dem onstrated that Pyk2 plays an im portant role in M AP kinase signalling cascades m ediated by elevation of intracellular Ca^"^, by activation of GPCRs, by stress signals or by PKC agonists (Dikic et al., 1996; Tokiwa et al., 1996). Activation of Pyk2 by bradykinin or LPA stimulates ERK activation by a mechanism involving Pyk2 autophosphorylation, association with Src, recruitment of a Grb2-Sos complex and subsequent activation of Ras (Dikic et a l, 1996; Lev et al., 1995).

In addition to the Nir proteins, several other intracellular proteins have been shown to interact with Pyk2. Specific structural motifs or tyrosine phosphorylation sites mediate these interactions. Recent studies have described the direct association of Pyk2 with the adapter protein Grb2, the tyrosine kinase Src and the ARF-GAP protein Pap (Pyk2 carboxy-terminal associated protein; Andreev et al., 1999; Dikic

et a l, 1996). Pap forms a stable complex with Pyk2 and is tyrosine phosphorylated upon Pyk2 activation. Pap is a multi domain protein that exhibits intrinsic GAP activity towards the small G proteins A rfl and Arf5 (Andreev et a l, 1999). O verexpression of Pap inhibits Golgi vesicle release. Recent studies have also demonstrated that Pyk2 directly interacts with the cytoskeletal proteins paxillin, p l3 0 “ * and Graf (Rac/Cdc42 GTPase-activating protein; Ohba et a l, 1998).

A lthough the role of Pyk2 in a variety of signalling cascades has been extensively studied, its actual physiological function rem ains largely unknown. Since Pyk2 is activated by a variety of extracellular stimuli in different cell types, it has been proposed that Pyk2 may facilitate cross talk between different intracellular signalling pathways (Lev et a l , 1999). Although phosphoinositides bind many actin- binding proteins and affect cell attachment, the role of the interaction between Pyk2 and N irl-2 has yet to be addressed. Based on the observations discussed earlier (Section 1.4) and genetic studies in D rosophila, Lev and colleagues have proposed that the rdgB proteins function in concert with Pyk2 and downstream of G-protein coupled receptors as com ponents of an evolutionarily conserved Ca^^ and phosphoinositide-dependent signalling (Lev et a l, 1999).

1.10 Summary

The Ptdlns-T P fam ily consists of the single domain yeast S e c l4 p and mammalian PITPa and PITP6 and the multi domain rdgB proteins. Numerous studies have indicated a requirem ent for Ptdlns-TPs during phospholipid m etabolism , phosphoinositide-mediated signalling and vesicle trafficking. As such, this family of proteins is thought to play a role in a variety of cellular processes. With the possible exception of yeast Secl4p, the actual biological functions of the Ptdlns-TPs remain to be characterised. Many basic questions remain essentially unresolved, including the structural basis for the recognition of specific phospholipids by the various Ptdlns-TPs and how the phospholipid binding and transfer activity of a Ptdlns-TP pertains to its in vivo function. Additionally, the existence of multiple Ptdlns-TPs raises the question of functional specificity and degeneracy between different Ptdlns- TP domains. At the start of this thesis it was hypothesised that the molecular and biochemical characterisation of the Ptdlns-TPs would provide a better understanding of the functional specificity and degeneracy within the Ptdlns-TP family. The following studies describe the cloning and characterisation of a novel member of the Ptdlns-TP family.