Richard A. Currie

Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities

The second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is generated by the action of phosphoinositide 3-kinase (PI 3-kinase), and regulates a plethora of cellular processes. An approach for dissecting the mechanisms by which these processes are regulated is to identify proteins that interact specifically with PtdIns(3,4,5)P(3). The pleckstrin homology (PH) domain has become recognized as the specialized module used by many proteins to interact with PtdIns(3,4,5)P(3). Recent work has led to the identification of a putative phosphatidylinositol 3,4,5-trisphosphate-binding motif (PPBM) at the N-terminal regions of PH domains that interact with this lipid. We have searched expressed sequence tag databases for novel proteins containing PH domains possessing a PPBM. Surprisingly, many of the PH domains that we identified do not bind PtdIns(3,4,5)P(3), but instead possess unexpected and novel phosphoinositide-binding specificities in vitro. These include proteins possessing PH domains that interact specifically with PtdIns(3,4)P(2) [TAPP1 (tandem PH-domain-containing protein-1) and TAPP2], PtdIns4P [FAPP1 (phosphatidylinositol-four-phosphate adaptor protein-1)], PtdIns3P [PEPP1 (phosphatidylinositol-three-phosphate-binding PH-domain protein-1) and AtPH1] and PtdIns(3,5)P(2) (centaurin-beta2). We have also identified two related homologues of PEPP1, termed PEPP2 and PEPP3, that may also interact with PtdIns3P. This study lays the foundation for future work to establish the phospholipid-binding specificities of these proteins in vivo, and their physiological role(s).

PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2

BACKGROUND Protein kinase B (PKB) is activated by phosphorylation of Thr308 and of Ser473. Thr308 is phosphorylated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1) but the identity of the kinase that phosphorylates Ser473 (provisionally termed PDK2) is unknown. RESULTS The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF). PIF is situated carboxy-terminal to the kinase domain of PRK2, and contains a consensus motif for phosphorylation by PDK2 similar to that found in PKBalpha, except that the residue equivalent to Ser473 is aspartic acid. Mutation of any of the conserved residues in the PDK2 motif of PIF prevented interaction of PIF with PDK1. Remarkably, interaction of PDK1 with PIF, or with a synthetic peptide encompassing the PDK2 consensus sequence of PIF, converted PDK1 from an enzyme that could phosphorylate only Thr308 of PKBalpha to one that phosphorylates both Thr308 and Ser473 of PKBalpha in a manner dependent on phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3). Furthermore, the interaction of PIF with PDK1 converted the PDK1 from a form that is not directly activated by PtdIns(3,4,5)P3 to a form that is activated threefold by PtdIns(3,4,5)P3. We have partially purified a kinase from brain extract that phosphorylates Ser473 of PKBalpha in a PtdIns(3,4,5)P3-dependent manner and that is immunoprecipitated with PDK1 antibodies. CONCLUSIONS PDK1 and PDK2 might be the same enzyme, the substrate specificity and activity of PDK1 being regulated through its interaction with another protein(s). PRK2 is a probable substrate for PDK1.

The PIF‐binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB

PKB/Akt, S6K1 and SGK are related protein kinases activated in a PI 3‐kinase‐dependent manner in response to insulin/growth factors signalling. Activ ation entails phosphorylation of these kinases at two residues, the T‐loop and the hydrophobic motif. PDK1 activates S6K, SGK and PKB isoforms by phosphorylating these kinases at their T‐loop. We demonstrate that a pocket in the kinase domain of PDK1, termed the ‘PIF‐binding pocket’, plays a key role in mediating the interaction and phosphorylation of S6K1 and SGK1 at their T‐loop motif by PDK1. Our data indicate that prior phosphorylation of S6K1 and SGK1 at their hydrophobic motif promotes their interaction with the PIF‐binding pocket of PDK1 and their T‐loop phosphorylation. Thus, the hydrophobic motif phosphorylation of S6K and SGK converts them into substrates that can be activated by PDK1. In contrast, the PIF‐binding pocket of PDK1 is not required for the phosphorylation of PKBα by PDK1. The PIF‐binding pocket represents a substrate recognition site on a protein kinase that is only required for the phosphorylation of a subset of its physiological substrates.

Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C‐terminal residues of PKA

The 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1) phosphorylates and activates a number of protein kinases of the AGC subfamily. The kinase domain of PDK1 interacts with a region of protein kinase C‐related kinase‐2 (PRK2), termed the PDK1‐interacting fragment (PIF), through a hydrophobic motif. Here we identify a hydrophobic pocket in the small lobe of the PDK1 kinase domain, separate from the ATP‐ and substrate‐binding sites, that interacts with PIF. Mutation of residues predicted to form part of this hydrophobic pocket either abolished or significantly diminished the affinity of PDK1 for PIF. PIF increased the rate at which PDK1 phosphorylated a synthetic dodecapeptide (T308tide), corresponding to the sequences surrounding the PDK1 phosphorylation site of PKB. This peptide is a poor substrate for PDK1, but a peptide comprising T308tide fused to the PDK1‐binding motif of PIF was a vastly superior substrate for PDK1. Our results suggest that the PIF‐binding pocket on the kinase domain of PDK1 acts as a ‘docking site’, enabling it to interact with and enhance the phosphorylation of its substrates.

DAPP1: a dual adaptor for phosphotyrosine and 3-phosphoinositides.

We have identified a novel 280 amino acid protein which contains a putative myristoylation site at its N-terminus followed by an Src homology (SH2) domain and a pleckstrin homology (PH) domain at its C-terminus. It has been termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1). DAPP1 is widely expressed and exhibits high-affinity interactions with PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2), but not with other phospholipids tested. These observations predict that DAPP1 will interact with both tyrosine phosphorylated proteins and 3-phosphoinositides and may therefore play a role in regulating the location and/or activity of such proteins(s) in response to agonists that elevate PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2).

Characterisation of a plant 3-phosphoinositide-dependent protein kinase-1 homologue which contains a pleckstrin homology domain

A plant homologue of mammalian 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1) has been identified in Arabidopsis and rice which displays 40% overall identity with human 3‐phosphoinositide‐dependent protein kinase‐1. Like the mammalian 3‐phosphoinositide‐dependent protein kinase‐1, Arabidopsis 3‐phosphoinositide‐dependent protein kinase‐1 and rice 3‐phosphoinositide‐dependent protein kinase‐1 possess a kinase domain at N‐termini and a pleckstrin homology domain at their C‐termini. Arabidopsis 3‐phosphoinositide‐dependent protein kinase‐1 can rescue lethality in Saccharomyces cerevisiae caused by disruption of the genes encoding yeast 3‐phosphoinositide‐dependent protein kinase‐1 homologues. Arabidopsis 3‐phosphoinositide‐dependent protein kinase‐1 interacts via its pleckstrin homology domain with phosphatidic acid, PtdIns3P, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 and to a lesser extent with PtdIns(4,5)P2 and PtdIns4P. Arabidopsis 3‐phosphoinositide‐dependent protein kinase‐1 is able to activate human protein kinase Bα (PKB/AKT) in the presence of PtdIns(3,4,5)P3. Arabidopsis 3‐phosphoinositide‐dependent protein kinase‐1 is only the second plant protein reported to possess a pleckstrin homology domain and the first plant protein shown to bind 3‐phosphoinositides.

Mdm2 binding to a conformationally sensitive domain on p53 can be modulated by RNA

Biochemical characterisation of the interaction of mdm2 protein with p53 protein has demonstrated that full‐length mdm2 does not bind stably to p53–DNA complexes, contrasting with C‐terminal truncations of mdm2 which do bind stably to p53–DNA complexes. In addition, tetrameric forms of the p53His175 mutant protein in the PAb1620+ conformation are reduced in binding to mdm2 protein. These data suggest that the mdm2 binding site in the BOX‐I domain of p53 becomes concealed when either p53 binds to DNA or when the core domain of p53 is unfolded by missense mutation. This further suggests that the C‐terminus of mdm2 protein contains a negative regulatory domain that affects mdm2 protein binding to a second, conformationally sensitive interaction site in the core domain of p53. We investigated whether there was a second docking site on p53 for mdm2 protein by examining the interaction of full‐length mdm2 with p53 lacking the BOX‐I domain. Although mdm2 protein did bind very weakly to p53 protein lacking the BOX‐I domain, addition of RNA activated mdm2 protein binding to this truncated form of p53. These data provide evidence for three previously undefined regulatory stages in the p53–mdm2 binding reaction: (1) conformational changes in p53 protein due to DNA binding or point mutation conceals a secondary docking site of mdm2 protein; (2) the C‐terminus of mdm2 is the primary determinant which confers this property upon mdm2 protein; and (3) mdm2 protein binding to this secondary interaction site within p53 can be stabilised by RNA.

The lipid transfer activity of phosphatidylinositol transfer protein is sufficient to account for enhanced phospholipase C activity in turkey erythrocyte ghosts

BACKGROUND The minor membrane phospholipid phosphatidylinositol 4, 5-bisphosphate (PIP2) has been implicated in the control of a number of cellular processes. Efficient synthesis of this lipid from phosphatidylinositol has been proposed to require the presence of a phosphatidylinositol/phosphatidylcholine transfer protein (PITP), which transfers phosphatidylinositol and phosphatidylcholine between membranes, but the mechanism by which PITP exerts its effects is currently unknown. The simplest hypothesis is that PITP replenishes agonist-sensitive pools of inositol lipids by transferring phosphatidylinositol from its site of synthesis to sites of consumption. Recent cellular studies, however, led to the proposal that PITP may play a more active role as a co-factor which stimulates the activity of phosphoinositide kinases and phospholipase C (PLC) by presenting protein-bound lipid substrates to these enzymes. We have exploited turkey erythrocyte membranes as a model system in which it has proved possible to distinguish between the above hypotheses of PITP function. RESULTS In turkey erythrocyte ghosts, agonist-stimulated PIP2 hydrolysis is initially rapid, but it declines and reaches a plateau when approximately 15% of the phosphatidylinositol has been consumed. PITP did not affect the initial rate of PIP2 hydrolysis, but greatly prolonged the linear phase of PLC activity until at least 70% of phosphatidylinositol was consumed. PITP did not enhance the initial rate of phosphatidylinositol 4-kinase activity but did increase the unstimulated steady-state levels of both phosphatidylinositol 4-phosphate and PIP2 by a catalytic mechanism, because the amount of polyphosphoinositides synthesized greatly exceeded the molar amount of PITP in the assay. Furthermore, when polyphosphoinositide synthesis was allowed to proceed in the presence of exogenous PITP, after washing ghosts to remove PITP before activation of PLC, enhanced inositol phosphate production was observed, whether or not PITP was present in the subsequent PLC assay. CONCLUSION PITP acts by catalytically transferring phosphatidylinositol down a chemical gradient which is created as a result of the depletion of phosphatidylinositol at its site of use by the concerted actions of the phosphoinositide kinases and PLC. PITP is therefore not a co-factor for the phosphoinositide-metabolizing enzymes present in turkey erythrocyte ghosts.