PH domain function

The observation that many PH domain-containing proteins are at some stage membrane- localised and the increasing amount of data on phosphoinositide-PH domain interactions (Lemmon et al., 1997), has supported the role of PH domains as membrane anchors.

One of the best characterised mechanisms in which a PH domain regulates membrane binding of a protein is that described for PLC 61. Immunolocalisation studies of overexpressed PLC 61 has demonstrated that the PH domain mediates the interaction of the enzyme with the plasma membrane (Paterson et al., 1995). Moreover, the specific recognition of PtdIns(4,5)P2 and D-Ins(l,4,5)Pg by the PH domain of PLC 61 has been shown to regulate the catalytic activity of the enzyme (Cifuentes et al., 1994; Ferguson et al., 1995; Garcia et al., 1995; Lemmon et al., 1995; Lomasney et al., 1996; Tall et al., 1997). Structural examination of full-length PLC 61 reveals that the PH domain is

connected to the rest of the enzyme by a flexible linker region (Essen et al., 1996). These results suggest a model whereby the PH domain, serving as a PtdIns(4,5)P2-specific tether, directs PLC 61 to membranes enriched in PtdIns(4,5)P2 permitting the active site to hydrolyse multiple substrate molecules before feedback inhibition by the polar head group product, D-Ins(l,4,5)P3.

Dbl, a DH domain containing protein, depends on the PH domain for membrane association and requires it to retain oncogenic activity (Zheng et al., 1996). A membrane targeting role has also been demonstrated for the N-terminal PH domain of Tiam l (Michiels et al., 1997). Tiam l, as well as Dbl homology and C-terminally adjacent PH domain, contains a PH domain in the N-terminal part of the protein. Using a detailed mutational strategy in combination with confocal laser scanning microscopy, it was shown that the N-terminal PH domain, and not the Dbl homology-adjacent PH domain, was essential for membrane localisation. The fact that the localisation was also serum induced and that the N-terminal PH domain could not be functionally exchanged for the PH domain of Dbl, the PH domain of pARK, or the C-terminal PH domain of Tiam l, argues for a specific membrane targeting function for this domain. Rapid endocytosis, mediated by dynamin 1 can be blocked by exogenous dynamin 1 PH domain (Artalejo et al., 1997). This inhibition could not be reproduced with the PtdIns(4,5)P2 binding PH domain of PLC 61 and was also isoform-specific for the PH domain of dynamin 1 mediated by two amino acids which are different between the PH domains of dynamin 1 and 2. A recombinant PH domain from Sos PH domain has been shown to localise to the plasma membrane in a serum-dependent manner (Chen et al., 1997). The Sos PH domain was found to bind PtdIns(4,5)P2 in vitro, however mutations in the Sos PH domain that reduced in vitro PtdIns(4,5)P2 binding did not affect serum dependent membrane localisation of the isolated domain. Both Artalejo et al., (1997) and Chen et al., (1997) surmised that the specific recognition by the dynamin 1 and Sos PH domains of a ligand other than PtdIns(4,5)P2 was responsible for membrane targeting. The PH domain of 1RS 1 is critical for effective signalling between the insulin receptor and 1RS 1 (Myers et al., 1995). Its deletion abolishes tyrosine phosphorylation of 1RS 1 and decouples it from downstream signalling (Yenush et al., 1996). Finally, guanine nucleotide exchange factors of the small G-protein ARFl also bind to membranes and phosphoinositide vesicles with their PH domains (Chardin et a l, 1996; Klarlund et a l, 1997).

The most compelling evidence for a specific cellular signalling role for PH domain recognition of phosphoinositides comes from studies of the Ser/Thr kinase Akt/PKB. Akt is activated when mammalian cells are stimulated with insulin and growth factors by a mechanism that requires the activation of PI 3-kinase (Burgering and Coffer, 1995;

Franke et al., 1995). Several groups have reported the binding of two PI 3-kinase products, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 to both intact Akt (James et al., 1996) and its isolated PH domain (Franke et al., 1997a; Freeh et al., 1997). However, the critical questions concerning the mechanism by which PI 3-kinase triggers the activation of Akt has only recently begun to be answered.

The current model is that growth factor induced stimulation of PI 3-kinase activity leads to an increase in the level of PtdIns(3,4,5)Pg/PtdIns(3,4)P2. This recruits Akt to the membrane where it is activated by phosphorylation at two residues, Thr 308 and Ser 473 (Alessi et al., 1996). This phosphorylation is thought to be mediated by two different kinases, one of which has recently been identified; PtdIns(3,4,5)Pg/PtdIns(3,4)P2 dependent kinase 1 (PDKl) (Alessi et al., 1997; Alessi et al., 1997a; Stokoe et al., 1997). In support of this signalling paradigm, membrane targeting of Akt (with its PH domain deleted) results in its enhanced ser/thr phosphorylation and activation in response to growth factor stimulation (Kohn et al., 1996).

Besides membrane targeting and phosphoinositide mediated cellular signalling, several PH domain containing proteins have also been associated with and even causative of human disease. For example, the oncogene product of Akt is overexpressed in a significant percentage of ovarian, pancreatic (Cheng et al., 1992; Cheng et al., 1996) and breast cancer cells (Jones et al., 1991). At present, the clearest example of a PH domain associated disease is X-linked agammaglobulinaemia (XLA). This disease is manifested as a B cell differentiation abnormality. Patients with XLA have decreased numbers of B lymphocytes and an almost complete lack of plasma cells, causing susceptibility to severe bacterial infections (Smith et al., 1994). The affected gene encodes a cytoplasmic protein tyrosine kinase, Bruton’s tyrosine kinase, Btk. A large number of mutations (>150) have been detected throughout Btk including several point mutations in the PH domain, that cause XLA (Vihinen et al., 1996). A Btk PH domain mutation Arg 28 to Cys is also the cause of a similar disease, xid, in mice (Rawlings et al., 1993). Recent studies have indicated that the isolated PH domain from Btk binds to Ins(l,3,4,5)P4 and PtdIns(3,4,5)Pg, and that mutations correlated with XLA specificity impair these interactions (Fukuda et al., 1996; Rameh et al., 1997, chapter 6). The molecular reasons for inactivation of Btk-mediated signal transduction by mutations in the PH domain will be discussed in subsequent chapters.