PROCESO DE CONSULTA (ENVÍO Y RECEPCIÓN DE

1.8.1 Protein phosphorylation

The reversible phosphorylation o f proteins represents a major mechanism for the control o f intracellular events in eukaryotic cells^^^^^’^^^^^. Processes as diverse as metabolism, contractility, membrane transport and secretion, the transcription and translation o f genes, cell division, fertilisation, and even memory, are all regulated by protein kinases and PP, which catalyse the opposing activities o f protein phosphorylation and dephosphorylation, respectively^^^^'^^^l The genes encoding a large number o f serine/threonine-specific, tyrosine-specific, or dual-specificity protein kinases have been cloned and compared, and it is estimated that hundreds o f novel protein kinases are yet to be identified^^^^l On the basis o f sequence conservation at the catalytic domain, it is clear that both protein serine/threonine kinases and tyrosine

kinases evolved from a common ancestor, then diverged to recognise distinct sets o f substrates, hence regulating distinct cellular responses^^^^l

Tyrosine phosphorylations play a major role in cell signalling by a number o f receptor systems, e.g. those for growth factors and interleukins, for antigen in T cells and B cells, and for immunoglobulins such as FceRI^^^^l PTK catalyze the transfer o f the y- phosphate o f ATP (or GTP) to the protein phenolic groups on tyrosine. PTK responsible for these phosphorylations are broadly classified into two groups; receptor PTK and non-receptor PTK, depending upon whether or not they possess extracellular ligand-binding domains. All receptor PTK have a large glycosylated, extracellular ligand-binding domain, a hydrophobic single transmembrane region, and a cytoplasmic region which incorporates the kinase catalytic domain^^^^l Non-receptor PTK have no extracellular domain, and are contained wholly within the cell, but some are associated with cell membranes and have been implicated in the signal transduction pathway as amplifiers o f the signal from the receptor PTK^^^^l

1.8.2 Protein dephosphorylation

Protein phosphorylations are counteracted by the action o f PP, structurally and functionally diverse enzymes represented by two distinct families. Serine/threonine PP dephosphorylate phosphoserine and phosphothreonine residues^^^’\ whereas the protein tyrosine phosphatases (PTP) dephosphorylate phosphotyrosines^^^^\

The most abundant protein serine/threonine phosphatases o f the eukaryotes are PPl, PP2A, PP2B, PP2C and the related mitochondrial pyruvate dehydrogenase phosphatase [263j,[264] PP2 PP2A are specifically and potently inhibited by a variety o f naturally occuring toxins such as okadaic acid (OA), a shellfish poison and microcystin, a liver toxin produced by blue algae^^^^l However, PP2B is only poorly inhibited by these toxins. Protein serine/threonine phosphatases are métallo enzymes and two divalent metal ions (Mn^^ and Fe^^ in P P l, Zn^^ and Fe^^ in PP2B) at the centre o f the catalytic

Despite extremely limited sequence similarity, all PTP share an active site motif consisting o f a cysteine and an arginine residue^^^^^. These phosphatases can be identified by their sensitivity to vanadate, insensitivity to OA, a lack o f dependence on metal ions, an ability to hydrolyse p-nitrophenyl phosphate (pNPP) and total loss o f activity upon mutation o f the active site cysteine to serine.

1.8.3 Role of PTK in immunological signal transduction of mast cells

Activation o f rat mast cells through the FcgRI requires a complex set o f interactions involving transmembrane subunits o f the FcgRI and two classes o f non-receptor PTK, the Src fajnily PTK p53/p56‘>'" (Lyn) and the Syk/ZAP family PTK (Syk)'“ *’.

The cytoplasmic domains o f the (5 and y subunits o f FcgRI contain a sequence consisting o f two YXXL/I cassettes separated by 6-8 amino acids^^^^\ This sequence, called IT AM, is also found in the cytoplasmic domains o f T and B cell receptors^^^^l The critical role o f IT AM in transducing signals was revealed by analysing the effect o f aggregating chimeric proteins possessing an intracellular ITAM-containing polypeptide fused to transmembrane and extracellular domains o f an unrelated protein. Aggregation o f such ITAM-containing fusion proteins was sufficient to induce both early and late signalling events normally observed upon clustering o f oligomeric receptors^^^*^. In mast cells, clustering o f FcgRI results in increased tyrosine phosphorylation and activation o f numerous signalling molecules, including the p and y chains o f FcgRI^^^^^, PTK o f the Src family (p56/p53'y” and ppbO'""")^^^^], Syk PTK^^'^'^^ PLC-y and mitogen activated protein (MAP) kinase^^^^^.

The rapid tyrosine phosphorylation o f FcgRI in the absence o f any intrinsic kinase activity o f the receptor suggested the presence o f closely associated tyrosine kinases. Additional data indicated that FcgRI was associated with Lyn kinase prior to receptor cross-linking and that Lyn initiated a cascade o f phosphorylation events in antigen- activated mast cells^^^^l These and other data led to the proposal o f a model for FcgRI function where the Lyn kinase is bound to the p subunit o f FCgRI and after FcgRI aggregation phosphorylates both p and y IT AM. The phosphorylated P IT AM serves to

anchor more Lyn, probably via interaction o f Lyn SH2 (Src homology 2)-ITAM phosphotyrosine, in the membrane domains containing aggregated FcgRI, whereas phosphorylated y ITAM serves as the docking site for Syk kinase, which is then phosphorylated and activated by Lyn, leading to downstream signal propagation^^^^^’^^^^l SH2 domains were first described on the basis o f a strong homology between members o f the Src tyrosine kinase family within their amino- terminal region, but have now been found in many proteins in signal transduction^^*®l They bind with high affinities to sequences that contain an upstream negative charge, followed within a few residues by a phosphorylated tyrosine^^*^^.

1.8.4 PTK and disease

As mentioned earlier, tyrosine kinases play a crucial role in many cell regulatory processes, and it is therefore not surprising that functional deviations o f PTK and PTP result in many diseases. An extensive amount o f work has been done on development o f potent and selective inhibitors for these two classes o f enzymes^^*^’^*"^^.

The first PTK, Rous sarcoma viral v-src oncoprotein, was discovered in

Since then, a large number o f viral and tumor oncogenes that encode PTK have been reported and many o f them have been implicated in human cancer^^^^l There are two major mechanisms of oncogenesis by these enzymes: (1) overexpression or amplification o f PTK as in the case o f the epidermal growth factor (EGF) receptor, Src and c-Yes and (2) expression o f mutated PTK either by chromosomal translocation (e.g. Trk) or mutation (e.g. Ret, Met) resulting in constitutively activated PTK through dimérisation or altered substrate specificity. Highly potent and selective inhibitors o f these PTK may have anti-cancer activity.

ZAP70 PTK is essential for T cell development and is activated downstream o f the T cell receptor. Mutational inactivation o f this enzyme causes severe combined immunodeficiency syndrom (SClD)^^**l Similarly, Btk non-receptor PTK is required for B cell development. Mutation in any SH3, SH2, or catalytic domain that leads to

leading to agammaglobulinemia^^^^l In non-insulin-dependent diabetes, there is an inactivating mutation at one or both alleles o f the insulin-receptor PTK.

Platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) and their receptors have been implicated in the formation o f atherosclerotic plaques^^^^^. Inhibitors o f these receptor PTK may be useful for the treatment o f restenosis. In psoriasis, the TGFa and /or amphiregulin genes are amplified, leading to the activation o f EGF receptor PTK^^^'l A potent EGF receptor PTK inhibitor may therefore be therapeutic for this disease.