PRINCIPIOS Y OBLIGACIONES EN GENERAL

1.4.4.1 Structure and specificity

The sequence homology betw een PH domains is less well conserved than that observed for SH2 and SH3 domains, resulting in the need for sensitive

database searching program s to identify novel members (Musacchio et al., 1993; Shaw, 1993; Gibson et al., 1994; Lemmon et al., 1996; Shaw, 1996). However, solution and crystal structures have revealed a remarkable

conservation of tertiary structure (Ferguson et al., 1994,1995), confirming that PH dom ains constitute independent protein modules. PH dom ains are

approxim ately 100-130 amino acids in length and m ay be split by inserted sequences- in the case of PLCy the PH dom ain is split by an insert of two SH3 dom ains and an SH2 domain.

N-term inal residues form two opposing P-sheets closed at one corner by an a - helix consisting of C-terminal residues (Ferguson et al., 1994, Shaw, 1996). The m ost variable face of the dom ain consists of positively charged loop regions betw een p-strands which in the case of PLCôl have been show n to bind

phosphatidylinositol 4,5-bisphosphate (PIP2) and inositol 1,4,5-triphosphate

(IP3) w ith high affinity (Ferguson et al., 1995) and in so doing may mediate m em brane localisation. Com parison of the Btk sequence w ith the crystal

structure of PLCôi complexed w ith IP3, suggest that residues m utated in XLA

patients (including the xid m utation) could be im portant for high affinity

binding to IP3 (Ferguson et al., 1995). Furthermore, a transform ing Btk E41K

CHAPTER 1__________________________________________________________ INTRODUCTION

m utation (section 1.7.3) is proposed to enable the form ation of strong bonds w ith phosphoinositides. As the Btk E41K m utant shows increased m em brane localisation (Li et al., 1995), these results provide circumstantial evidence that the Btk PH dom ain functions to localise Btk to the m em brane by binding m em brane lipids (see below). However, the high affinity of the PLCôi PH

dom ain for IP3 m ay not be representative of a function for all PH domains,

m any of which have low affinities for lipid ligands (Ferguson et al., 1995). Recently, structures of the phosphotyrosine binding (PTB) dom ains of She and IRS-1 have revealed that these are members of a distinct class of PH domain, sharing the p-sandw ich/ a-helix structure but recognising phosphotyrosine residues in the context of xNPxpY (Eck et al., 1996; Lemmon et al., 1996).

1.4.4.2 Function

In contrast to the well defined ligands for SH2 and SH3 domains, several potential candidates exist for PH domains. Indeed, the diversity between sequences of PH dom ains may reflect diverse functions and ligands. PH dom ains are found in a variety of cytoskeletal and signalling proteins including pleckstrin and actin filament associated proteins (AFAP-110 and 120); several serin e/ threonine kinases; Ras GTPase activating protein

(RasGAP); several GEFs including mSos and Vav; dynamin; IRS-1 and several PLC p, y and Ô isoforms (Shaw, 1996). Members of the Btk/Tec family are the only tyrosine kinases that contain PH dom ains (Shaw, 1993).

Many PH dom ain containing molecules require m em brane localisation for function. Suggestions that the PH dom ains provides this function are given by studies that show the PH dom ain is required for the transform ing ability of the GEF, Lfc and that it can be functionally replaced by a m em brane localisation signal (reviewed in Shaw, 1996).

Interestingly, a point m utation in the PH dom ain of neural W iskott-Aldrich syndrom e protein (N-WASP, section 1.10.2) abolished its association with

CHAPTER 1__________________________________________________________ INTRODUCTION

actin and led to nuclear localisation (Miki et aL, 1996). This correlated w ith a

decrease in the ciffinity of the m utant PH dom ain for PIP2.

Btk/Tec family kinases lack m yristylation signals but appear to translocate to the m em brane in a signal dependent fashion requiring a functional PH

dom ain (Kawakami et aL, 1994; Li et aL, 1995). The importance of the Btk PH dom ain for Btk function is highlighted by the fact that the xid m utation in the PH dom ain or a total absence of Btk result in the same phenotype (Kerner et aL, 1995; Khan et aL, 1995).

Recently, the Btk PH dom ain has been show n to have a strong in vitro

specificity for phosphatidylinositol 3,4,5-triphosphate (PIP3) (Salim et aL,

1996), suggesting that Btk function m ay be linked to that of PI3 kinase. This is discussed further in sections 7.2.2 and 7.4.2.

Two possibilities other than (or in addition to) putative m em brane lipid binding by the PH dom ain could account for m em brane localisation of Btk. Firstly, both the Btk and p-adrenergic kinase PH dom ains have been

implicated in binding to the Py subunits of heterotrimeric G-proteins (Koch et aL, 1993; Tsukada et aL, 1994). However, the fusion proteins used in both studies contained sequences C-terminal to the PH domains. Indeed binding of Py subunits did not require the first 68 amino acids of the Btk PH domain. Thus the contribution of the PH dom ain has not been fully determined. How ever the interaction remains intriguing in light of the finding that Gpy subunits can stimulate Btk kinase activity (section 1.7.4) (Langhans

Rajasekaran et aL, 1995). Secondly, the Btk PH dom ain has been show n to m ediate the in vivo association of Btk w ith protein kinase C (PKC)p in m urine m ast cells (Yao et aL, 1994). In addition to phosphorylating and dow n

regulating Btk, PKC phosphorylates other PH dom ain containing molecules including pleckstrin and dynamin. It has been proposed that these interactions may be m ediated through m em brane associated RACK (receptor for active PKC) w hich contains WD-40 repeats found in m any G protein p subunits (Shaw, 1996).

CHAPTER 1__________________________________________________________ INTRODUCTION

Recently, sequence homology has suggested the presence of a PH dom ain at the N term inus of WASP (Miki et al., 1996). Thus m utations in PH domains have been show n to give rise to two immunodeficiencies, WAS and XLA (Schwarz et al., 1996; Vihinen et al., 1996), underscoring their importance in haem atopoietic signal transduction.