RESUMO
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).
Assuntos
Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Fosfatidilinositóis/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Clonagem Molecular , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Plantas , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Ressonância de Plasmônio de SuperfícieRESUMO
Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP3) play an essential role in the regulation of neutrophil functions by the chemoattractant formylmethionyl-leucylphenylalanine (FMLP). Here we show that permeabilization of human neutrophils leads to loss of cytosolic components, including PI3Kgamma, and causes the loss of FMLP-dependent production of PIP3. FMLP-sensitive synthesis of PIP3 could be restored by reconstitution of permeabilized neutrophils with recombinant PI3Kgamma. Admixture of recombinant phosphatidylinositol transfer protein (PITP) to the reconstitution cocktail produced a further increase of PIP3 synthesis, whereas pertussis toxin suppressed the FMLP-dependent production of PIP3. We conclude that FMLP-sensitive PIP3 formation in human neutrophils involves the FMLP receptor, heterotrimeric G-proteins of the Gi type, PI3Kgamma and PITP.