DE LAS INFRACCIONES Y SANCIONES ADMINISTRATIVAS

MAP kinase pathw ays are ubiquitous serine/ threonine kinase cascades that couple small GTPases such as Ras, Rho and Cdc42Hs to specific nuclear transcription events (Seger and Krebs, 1995; Su and Karin, 1996). The best defined pathw ay is the R as/R af/M E K /E rk 1,2 pathw ay in which Erk 1 and 2 are MAP kinases (Fig.1.8). While MAP kinases phosphorylate m icrotubule associated protein (MAP) they also phosphorylate and activate num erous transcription factors such as c-Jun, c-Fos, c-Myc and Elk-1, inducing mitogenic responses.

Erks 1 and 2 are stim ulated following BCR cross-linking in mouse and hum an cell lines as dem onstrated by tyrosine phosphorylation and an increase in its serine/threonine kinase activity tow ard MAP-2 (Casillas et aL, 1991;

Sutherland et aL, 1996). A similar response is seen in T cells following stim ulation of the TCR (Ettehadieh et aL, 1992). The upstream elements Ras,

CHAPTER 1__________________________________________________________ INTRODUCTION

Raf, MEK, Erk 1 /2 are all stimulated as show n by an increase in GTP bound Ras (H arwood and Cambier, 1993; Tordai et aL, 1994) and an increased kinase activity of Raf, MEK and Erk (Tordai et aL, 1994). The MARK substrate p90'^®’^ is also phosphorylated in response to BCR (Tordai et aL, 1994) as is RasGAP (Gold et aL, 1993). BCR stim ulation of the m urine WEHI-231 cell line also causes activation of another class of MAP kinases, fu n N -term inal kinases (JNKs) implicated in signalling through Rho family GTPases, but only a minimal activation of the p38 MAP kinase (Sutherland et aL, 1996).

She becomes tyrosine phosphorylated and inducibly binds to G rb2/ mSos following BCR cross-linking in a variety of m urine and hum an B cells. The resulting S h e/G rb 2 / mSos complexes are translocated to the m em brane (Ravichandran et aL, 1993; Saxton et aL, 1994), although it is unclear how they interact w ith the BCR (Ravichandran et aL, 1993; Osm an et aL, 1995). By analogy w ith G rb2/ mSos function in GFR signalling (Buday and Downward, 1993), translocation of mSos to the m em brane may function by bringing it into close proximity w ith its target, Ras, leading to GTP uptake. GTP bound Ras is then capable of stim ulating Raf, initiating the Erk pathw ay (Avruch et aL, 1994) (Fig. 1.8).

It is tem pting to speculate that such events depend on the form ation of a clustered tyrosine kinase complex (Fig. 1.8). Syk is necessary for the

phosphorylation of She and MAP kinase (Richards et aL, 1996) and both Syk and She are precipitated by dp IT AMs (Johnson et aL, 1995). In such

complexes, Syk autophosphorylation may generate binding sites for SH2 and phosphotyrosine binding dom ain (PTB) dom ain containing molecules and induce a variety of effector pathways. Notably, Syk inducibly associates w ith a 145 kDa phosphoprotein following BCR stim ulation (Crowley et aL, 1996) and phosphorylated ZAP-70 is bound by the SH2 dom ain of Vav following TCR stim ulation (Campbell et aL, 1995).

CHAPTER 1___________________________________________________________INTRODUCTION

1.7.6 Vav

The product of the V A V proto-oncogene is a haematopoietically restricted

protein containing an SH2 and tw o SH3 dom ains as well as a region w ith homology to the Rho family GEF, Dbl (Hu et al., 1993; Khosravi Far et al., 1994). Vav is tyrosine phosphorylated in response to BCR stim ulation (Bustelo and Barbacid, 1992) and m ature B cells from VA V knockout mice do not respond to BCR cross-linking (Zhang et al., 1995; Tarakhovsky et al., 1995). Vav associates w ith tyrosine phosphorylated SLP-76 following TCR

stim ulation (Weng et al., 1994) and also w ith the C rb 2 like adapter molecule Crk (Smit et al., 1996).

Fibroblasts transform ed w ith Vav exhibit a phenotype similar to those

transform ed w ith Dbl but distinct from those transform ed w ith Ras, including the appearance of stress fibres and focal adhesions (Khosravi Far et al., 1994). It is therefore possible that Vav m ay couple the activation of Rho family proteins to the BCR (Fig.1.8). Recent data suggests that CD19 m ay be im portant for Vav activation and the subsequent stim ulation of Rho and dow nstream JNKs (D. Fearon, personal communication).

1.7.7 PI3 kinase

The p 8 5 /p llO PI3 kinase (Smit et aL, 1996) is a lipid kinase that preferentially

phosphorylates PIP2 upon the 3 position of the inositol ring. PI3 kinase

associates w ith stim ulated CFRs and is required for mitogenic responses (Valius and Kazlauskas, 1993).

P13 kinase is tyrosine phosphorylated and stim ulated following BCR cross- linking (Cold et al., 1992; Yamanashi et al., 1992; Pleiman et al., 1994; Beckwith et al., 1996) perhaps by binding the SH3 dom ains of Fyn and Lyn which can stimulate PI3-kinase activity in vitro (Pleiman et al., 1994). Furtherm ore, BCR induced activation of P13 kinase can be inhibited by proline rich peptides predicted to disrupt this association (Pleiman et al., 1994), suggesting a novel

CHAPTER 1__________________________________________________________ INTRODUCTION

link betw een BCR stim ulation and cellular responses m ediated by SH3

domains. The p85 subunit of PI3 kinase contains an SH2 dom ain which binds to a phosphorylated YxxM motif in the tail of CD19 (Tuveson et al., 1993; Chalupny et al., 1995) following mIgM or CD19 cross-linking. This also causes stim ulation of PI3 kinase activity (Tuveson et al., 1993). As co-ligation of CD19 w ith mIgM increases the sensitivity of B cells to mIgM stim ulation (Carter and Fearon, 1992) it is possible that CD19 binding brings PI3 kinase a) into close proxim ity to receptor bound Src kinases and b) into close proximity to its

substrate PIP2 in the cell membrane. It has recently been show n that

m em brane targeted forms of pllO are sufficient to stimulate dow nstream responses, including JNK activation, in the absence of grow th factor stim ulation (Klippel et aL, 1996).

A potential link betw een P13 kinase activity and cellular responses has been suggested by the observation that its lipid product, phosphatidyl 3,4,5-

triphospliate (PIP3), can activate purified PKCÇ (Nakanishi et al., 1993), which

in tu rn has been implicated in the control of the transcription factor NF-kB (Diaz Meco et al., 1993). PI3 kinase has also been show n to interact w ith Ras

CdicA-irtà

(Hu et al., 1995), Rac and-Rh©’ (Marshall, 1995). O ther potential functions of PI3 kinase may involve the induction of m em brane ruffling (W ennstrom et al., 1994), glucose uptake (Smit et al., 1996) (Fig.1.8) and m em brane localisation of PH dom ain containing proteins (section 7.7.2).

The im portance of PI3 kinase activity in B cell signalling has been dem onstrated using the specific inhibitor, w ortm annin. W ortm annin

treatm ent of hum an B lym phom a cells inhibits the ability of BCR cross-linking to cause grow th inhibition (Beckwith et al., 1996). This effect occurred despite norm al tyrosine kinase and PKC activation suggesting PI3 kinase lies

dow nstream or independent of tyrosine kinase activity.

A related family of enzymes including D N A -dependent protein kinase (DNA- PK) and the product of the ataxia telangiectasia gene (ATM) possess both PI3 kinase and protein kinase activities. They are believed to function in double

CHAPTER 1__________________________________________________________ INTRODUCTION

stranded DNA repair (notably following haematopoietic cell recombination events) and although they have not been directly implicated in BCR

signalling, DNA-PK and ATM protein m utations lead to a m urine form of SCID and the hum an immunodeficiency, ataxia telangiectasia, respectively (Abraham, 1996).

1.7.8 H S l

H Sl is a 75 kDa protein th at contains a C-terminal SH3 dom ain and a helix- loop-helix motif found in the Jun, Fos and Myc transcription factors. It is tyrosine phosphorylated in response to BCR cross-linking of D audi cells and inducibly associates w ith Lyn (Yamanashi et al., 1993). Its function is unclear but it is essential for the apoptotic response of WEHI-231 cells (Fukuda et al., 1995).

1.7.9 The cytoskeleton

C urrent know ledge of the role of the cytoskeleton in B cell signalling is

extremely limited. However, m any inferences can be taken from other systems which suggest it is a crucial effector of signal transduction .

Recent reports show association of the TCRÇ chain w ith the cytoskeleton via actin microfilaments. One of these (Caplan et al., 1995) dem onstrates the association in resting T cells while another, more detailed study shows that it occurs in response to TCR cross-linking on m ature but not im m ature

thymocytes and requires the presence of an intact IT AM (Rozdzial et al., 1995). The finding th at cytoskeletal association of TCRÇ correlated w ith IL-2

production is in keeping w ith findings that some receptor tyrosine kinases have higher affinity for their ligands w hen bound to the cytoskeleton . BCR cross-linking results in actin polymerisation (Melamed et al., 1991) and Syk is required for filamentous (F)-actin form ation in response to clustering of IT AM containing chimeras (Cox et al., 1996). The mechanisms that lay behind

CHAPTER 1__________________________________________________________ INTRODUCTION

this rem ain unclear. Im portantly, capping of m ig and co-capping of m ig w ith Ras is prevented by the cytoskeletal disrupters cytochalasin D and colchicine suggesting a functional role for the cytoskeleton in signal transduction.

Src family tyrosine kinases have been show n to have a role in organising the cytoskeleton through phosphorylation of cytoskeletal com ponents (Thomas et al., 1995) and m ay also regulate the activity of Fak (Schlaepfer et al., 1994). Also, SH3 dom ains have been show n to direct protein localisation to the cytoskeleton (Abraham, 1996) and so may represent a link betw een m any BCR associated molecules and cytoskeletal changes.

The involvem ent of the Rho family of small GTP binding proteins in

regulating the cytoskeleton is well docum ented (Nobes and Hall, 1995). The recent discovery that WASP binds active Cdc42Hs (Symons et al., 1996; A spenstrom et al., 1996) has implicated WASP as an effector for this Rho family member. The importance of WASP in norm al im m une function is highlighted by num erous im m une defects (including cytoskeletal

abnormalities) in patients w ith W iskott-Aldrich syndrom e (WAS) w ho express m utant or no protein (see below). A putative link betw een BCR signalling and WASP is discussed in C hapter 6.

1.8

BTK IS IMPLICATED IN SIGNALLING