Phospholipase C <i>δ</i>1 requires a pleckstrin homology domain for interaction with the plasma membrane

Paterson, Hugh; Savopoulos, John; Perišić, Olga; Cheung, Robert; Ellis, Moira V.; Williams, Roger L.; Katan, Matilda

doi:10.1042/bj3120661

Cited by 119 publications

(103 citation statements)

References 13 publications

(26 reference statements)

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“…1 C). It was reported that the δ1 isozyme is located in the cytosol and in membrane fractions [33,35,36], whereas the δ4 isozyme was found exclusively in the nucleus [22]. The results (Fig.…”

Section: Resultssupporting

confidence: 55%

Localization of phospholipase C δ3 in the cell and regulation of its activity by phospholipids and calcium

Pawełczyk

Matecki

1998

European Journal of Biochemistry

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The localization of phospholipase C δ3 (PLC δ3) in the cell and its regulatory properties has been investigated. Western blotting showed that human platelet PLC δ3 is located in the membrane and cytosolic fraction. The enzyme amount in the cytosolic fraction was significantly lower than that in the membrane fraction. In rat liver, PLC δ3 was present in both the membrane and cytosolic fraction and was absent in nuclei. Examination of the effects of phospholipids on PLC δ3 revealed that this enzyme is inhibited by phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). Similar inhibition was observed in the presence of sphingomyelin and phosphatidylserine (PtdSer). This is in contrast to PLC δ1, which is activated by PtdCho and PtdEtn. In a detergent assay, PLC δ1 is activated by spermine and sphingosine, whereas PLC δ3 was inhibited by both these compounds at concentrations that maximally stimulated PLC δ1. A deletion mutant of PLC δ3, lacking the entire pleckstrin homology (PH) domain (residues 1Ϫ137), was fully active in the detergent assay, and it was inhibited by spermine, sphingosine and phospholipids to the same extent as the native enzyme. PLC δ3 activation required calcium ions. The relationship between the Ca 2ϩ concentration and enzymatic activity was almost identical for the deletion mutant and the native enzyme. However, in the liposome assay, PLC δ3 was less sensitive to Ca 2ϩ stimulation. This is in contrast to PLC δ1, which is equally sensitive to Ca 2ϩ stimulation in both the detergent and liposome assays. We conclude that Ca 2ϩ is necessary to induce specific conformational changes of PLC δ3, which leads to a productive orientation of the catalytic domain relative to the membrane. The regulatory properties of PLC δ3 described in this report suggest that PLC δ3 has a relatively low activity in cellular conditions that fully activate PLC δ1.Keywords : phospholipase C δ3; platelets; localization; phospholipids ; calcium.Phospholipase C (PLC) hydrolyzes phosphatidylinositol located in cellular membranes to generate diacylglycerol and inositol 1,4,5-triphosphate (InsP 3 ). Diacylglycerol activates protein kinase C and InsP 3 mobilizes Ca 2ϩ from intracellular stores [1,2]. Changes in Ca 2ϩ and diacylglycerol levels affect virtually every aspect of cellular regulation directly or indirectly. The control of PLC activity is thus one of the major starting points for cellular regulation.To date, three major types of phosphoinositide-specific phospholipase C species named β, γ and δ, have been characterized [3,4]. Each type of PLC is regulated differently. PLC γ appears to be regulated by tyrosine phosphorylation in response to growth factor receptor occupancy [5]. The phosphorylation of PLC γ does not affect the kinetic properties of the enzyme, but causes a redistribution of the enzyme from the cytosol to cell membrane [6,7]. Tyrosine phosphorylation of PLC γ promotes its association with actin components of the cytoskeleton [8,9]. PLC β isozymes are activated by the A and βγ subunits of the h...

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Section: Resultssupporting

confidence: 55%

Localization of phospholipase C δ3 in the cell and regulation of its activity by phospholipids and calcium

Pawełczyk

Matecki

1998

European Journal of Biochemistry

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“…These ®ndings suggest that the second step is regulated by binding of PI3-K-generated D3 PPIs to the PH domain. The membrane anchoring of PH domain-containing proteins via the interaction of the PH domain with membrane lipids has been suggested for several proteins including PLC-d1 (Paterson et al, 1995), SOS (Chen et al, 1997), IRS-1 (Yenush et al, 1996) and pleckstrin .…”

Section: Discussionmentioning

confidence: 99%

Akt activation by growth factors is a multiple-step process: the role of the PH domain

et al. 1998

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The protein kinase encoded by the Akt proto-oncogene is activated by phospholipid binding, membrane translocation and phosphorylation. To address the relative roles of these mechanisms of Akt activation, we have employed a combination of genetic and pharmacological approaches. Transient transfection of NIH3T3 cells with wild-type Akt, pleckstrin homology (PH) domain mutants, generated on the basis of a PH domain structural model, and phosphorylation site Akt mutants provided evidence for a model of Akt activation consisting of three sequential steps: (1) a PH domain-dependent, growth factor-independent step, marked by constitutive phosphorylation of threonine 450 (T450) and perhaps serine 124 (S124), that renders the protein responsive to subsequent activation events; (2) a growth factor-induced, PI3-K-dependent membrane-translocation step; and (3) a PI3-K-dependent step, characterized by phosphorylation at T308 and S473, that occurs in the cell membrane and is required for activation. When forced to translocate to the membrane, wild-type Akt and PH domain Akt mutants that are defective in the ®rst step become constitutively active, suggesting that the purpose of this step is to prepare the protein for membrane translocation. Both growth factor stimulation and forced membrane translocation, however, failed to activate a T308A mutant. This, combined with the ®nding that T308D/S473D double mutant is constitutively active, suggests that the purpose of the three-step process of Akt activation is the phosphorylation of the protein at T308 and S473. The proposed model provides a framework for a comprehensive understanding of the temporal and spatial requirements for Akt activation by growth factors.

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“…Many isolated PH domains have been shown to have high anity for phosphoinositides or inositolphosphates and it has, therefore, been proposed that the function of PH domains is to localize proteins to the plasma membrane (Shaw, 1996). Indeed, in the case of phospholipase C delta 1 (Paterson et al, 1995), human Actin Onco-Lbc Figure 4 Onco-Lbc is localized along actin stress ®bers. Serum-starved Swiss 3T3 cells were microinjected with 0.1 mg/mL oncoLbc plasmid DNA, allowed to express protein for 4 h then ®xed and stained with TRITC-conjugated phalloidin (left panel) and anti-FLAG monoclonal antibody and FITC-conjugated donkey anti-mouse antibody (right panel) NIH3T3 cells were seeded at 1.3610 5 cells/100 mm dish in DMEM and 10% newborn calf serum, the next day, 0.025 ± 1 mg of plasmid was transfected with 15 mg of high molecular weight NIH3T3 carrier DNA.…”

Section: Resultsmentioning

confidence: 99%

Distinct roles for DH and PH domains in the Lbc oncogene

et al. 1997

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Members of the Dbl-homology (DH) family of proteins promote guanine nucleotide exchange on Rho GTPases. Lbc, which speci®cally acts on Rho but not Rac or Cdc42, was isolated as a transforming oncogene and is composed of a DH and a Pleckstrin-homology (PH) domain. We show here that the Lbc DH domain alone is capable of stimulating new DNA synthesis in quiescent ®broblasts and Rho-dependent actin stress ®ber assembly. However, the PH domain is required for subcellular localization of Lbc along actin stress ®bers and for ecient transformation of NIH3T3 cells. The results show that, in contrast to other Dbl-homology proteins, the PH domain of Lbc is dispensable for activation of Rho in vivo. The PH domain-dependent subcellular localization of Lbc may, however, be important for growth factor activation of endogenous Lbc and for oncogenic transformation.

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Phospholipase C δ1 requires a pleckstrin homology domain for interaction with the plasma membrane

Cited by 119 publications

References 13 publications

Localization of phospholipase C δ3 in the cell and regulation of its activity by phospholipids and calcium

Localization of phospholipase C δ3 in the cell and regulation of its activity by phospholipids and calcium

Akt activation by growth factors is a multiple-step process: the role of the PH domain

Distinct roles for DH and PH domains in the Lbc oncogene

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