Cloning and Identification of Amino Acid Residues of Human Phospholipase Cδ1 Essential for Catalysis

Cheng, Hwei‐Fang; Jiang, Meei Jyh; Chen, Chih-Lin; Liu, Su-Min; Wong, Li Ping; Lomasney, Jon W.; King, Klim

doi:10.1074/jbc.270.10.5495

Cited by 79 publications

(81 citation statements)

References 44 publications

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“…A conserved structure of all squalene synthases is an α-helical core surrounding an active site, containing signature 'aspartate-rich' sequences [2]; sea urchin squalene synthase has homology to known protein sequences, particularly in regions interacting with prenyl substrates [71]. Domains critical for PLC activity are found in urchin PLC: sea urchin PLC-δ contains Ser522, Lys438, Lys440, and Arg549 that are important for human PLC activity [19,33]. Based on structural/functional studies of human and sea urchin PI-3K, domains involved in catalysis (phox homology (PX) and C2 domains) are quite similar and likely mediate similar protein-protein interactions and/or binding to membrane lipids [22,73].…”

Section: Ex Vivo Incubations Target Enzymatic Pathwaysmentioning

confidence: 99%

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

Rogasevskaia

Coorssen

2011

J Chem Biol

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Studies using isolated sea urchin cortical vesicles have proven invaluable in dissecting mechanisms of Ca 2+ -triggered membrane fusion. However, only acute molecular manipulations are possible in vitro. Here, using selective pharmacological manipulations of sea urchin eggs ex vivo, we test the hypothesis that specific lipidic components of the membrane matrix selectively affect defined late stages of exocytosis, particularly the Ca 2+ -triggered steps of fast membrane fusion. Egg treatments with cholesterol-lowering drugs resulted in the inhibition of vesicle fusion. Exogenous cholesterol recovered fusion extent and efficiency in cholesterol-depleted membranes; α-tocopherol, a structurally dissimilar curvature analogue, selectively restored fusion extent. Inhibition of phospholipase C reduced vesicle phosphatidylethanolamine and suppressed both the extent and kinetics of fusion. Although phosphatidylinositol-3-kinase inhibition altered levels of polyphosphoinositide species and reduced all fusion parameters, sequestering polyphosphoinositides selectively inhibited fusion kinetics. Thus, cholesterol and phosphatidylethanolamine play direct roles in the fusion pathway, contributing negative curvature. Cholesterol also organizes the physiological fusion site, defining fusion efficiency. A selective influence of phosphatidylethanolamine on fusion kinetics sheds light on the local microdomain structure at the site of docking/fusion. Polyphosphoinositides have modulatory upstream roles in priming: alterations in specific polyphosphoinositides likely represent the terminal priming steps defining fully docked, release-ready vesicles. Thus, this pharmacological approach has the potential to be a robust high-throughput platform to identify molecular components of the physiological fusion machine critical to docking, priming, and triggered fusion.

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Section: Ex Vivo Incubations Target Enzymatic Pathwaysmentioning

confidence: 99%

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

Rogasevskaia

Coorssen

2011

J Chem Biol

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“…To express PLC␦1 under the control of the T7 promoter, the coding sequence for PLC␦1 was cloned into pRSETA. The resulting expression construct (pRSETAplc) was transformed into the Escherichia coli strain BL21(DE3)pLys (Novagen), and the protein was isolated and purified as described previously (20). Phosphatidylethanolamine, PA, PC, and PS were obtained from Avanti Polar Lipids Inc. PIP 2 and dodecyl maltoside was obtained from Calbiochem.…”

Section: Methodsmentioning

confidence: 99%

“…Centrifugation Binding Assay-The binding of PLC␦1 to phospholipid vesicles was estimated by a centrifugation assay (20,23). The free Ca 2ϩ concentration was calculated according to Fabiato and Fabiato (24).…”

Section: Methodsmentioning

confidence: 99%

“…In the C2 loop deletion mutant (⌬646 -654), residues 646 -654 implicated in the divalent metal binding were deleted. These mutant forms of PLC␦1 were constructed, expressed, and purified as described previously (20,21).…”

Section: Methodsmentioning

confidence: 99%

“…Phospholipid Binding Assay-Phospholipid vesicles composed of PS/PC or PA/PC mixtures were prepared as described by Mueller et al (22) with slight modifications (20,21). A dry phospholipid film was formed by slowly blowing 0.25 ml of chloroform/methanol (2:1 v/v) containing mixed lipids (300 nmol or the indicated concentration of each of the indicated phospholipids) under a stream of nitrogen followed by freeze-drying under vacuum for 4 h. The phospholipid film was hydrated under nitrogen with 0.5 ml of nitrogen-aerated 0.18 M sucrose for 18 h at 4°C followed by mixing with an equal volume of distilled H 2 O. Vesicles were isolated from the pellet by centrifuging the hydrated phospholipids at 1200 ϫ g for 20 min.…”

Section: Methodsmentioning

confidence: 99%

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Activation of Phospholipase C δ1 through C2 Domain by a Ca2+-Enzyme-Phosphatidylserine Ternary Complex

Lomasney¹,

Cheng²,

Roffler³

et al. 1999

Journal of Biological Chemistry

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The concentration of free Ca 2؉ and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C ␦1 (PLC␦1). The rate of PLC␦1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated 20-fold by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca 2؉ concentration required for half-maximal activation of PLC␦1 from 5.4 to 0.5 M. Approximately 12 distinct isoforms of phospholipase C catalyze the Ca 2ϩ -dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 1 to yield the second messengers inositol trisphosphate (IP 3 ) and diacylglycerol (1, 2). This constitutes one of the major pathways for receptor-coupled signaling at the plasma membrane of most eucaryotic cells. Three families of PLC isoforms have been described in mammals: PLC␤, PLC␦, and PLC␥ (2). The members of each family are highly homologous to one another at the amino acid sequence level, but little identity exists between members of different families (2). Three exceptions to this divergence in structure are the catalytic domain, the C2 domain, and the N-terminal pleckstrin homology (PH) domain (1, 3).Among the initial steps in activation of PLC is a translocation to the plasma membrane. The enzyme binds to the lipidwater interface via the noncatalytic lipid binding PH domain, which is located near the N terminus of PLC␦1 and binds multiple phosphoinositides such as PIP 2 and IP 3 (4 -9). This domain allows the enzyme to catalyze the hydrolysis of many substrate molecules without falling off the interface, a process referred to as processive catalysis (5, 9). Because PH domain binds IP 3 tightly, it also could function as a feedback regulator of catalysis. Although much is known about the function of the PH domain in PLC␦1, very little is known about another lipid binding motif, the C2 domain. The C2 domain comprises approximately 130 amino acid residues and has been found in nearly 100 signaling molecules (10). The C2 domain was first identified in protein kinase C, and its function was implicated in Ca 2ϩ -dependent phospholipid interactions (11). Structural studies estimate three to four divalent metal binding sites in the C2 domain of PLC␦1 (12). The C2 domain in the C terminus of PLC␦1 is essential for catalysis, because partial deletion of the C2 domain results in an inactive enzyme (13). However, the molecular mechanism by which C2 domain functions in PLC␦1 catalysis still awaits further investigation.PLC is a prototype for enzymes that operate at an interface. As for other membrane-associated enzymes, the ability of PLC to catalyze the hydrolysis of PI or PIP 2 is influenced remarkably by the presence of nonsubstrate phospholipids in the membrane (14 -19). The molecular mechanisms by which nonsubstrate phospholipid affects PLC activity is largely unknown. Nonsubstrate phospholipids could alter the structure of the PLC-membrane interface or the net charge of the interface or could promote a ...

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Cytogenetic and molecular studies of a familial paracentric inversion of Y chromosome present in a patient with ambiguous genitalia

Liou

Gibson

et al. 1997

Am. J. Med. Genet.

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Here we describe the first reported case of a patient with a familial paracentric inversion in the long arm of the Y chromosome and ambiguous genitalia. FISH analyses with Y chromosome YACs demonstrated that the inversion breakpoints of the patients and the father's Ys appear to be the same and lie within interval 5B of the Y chromosome. PCR and sequence analysis indicated that our patient carries a normal SRY gene. For an additional comparison of the patient's inv(Y) with the father, two other Y chromosome sequences were examined. Molecular studies of this familial inverted Y chromosome showed no differences in the ZFY and TSPY genes between the father and the patient suggesting that the short arm of our patient's inv(Y) is identical to that of the patient's father. Southern analysis using a probe of the DAX-1 gene indicated that a single copy of DSS (dosage sensitive sex reversal) locus was present in the patient. Our results suggest that the abnormal sexual development in our patient is likely attributable to (an)other mechanism(s) than mutation in the SRY gene and dosage alteration of the DAX-1 gene.

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Cloning and Identification of Amino Acid Residues of Human Phospholipase Cδ1 Essential for Catalysis

Cited by 79 publications

References 44 publications

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

Activation of Phospholipase C δ1 through C2 Domain by a Ca2+-Enzyme-Phosphatidylserine Ternary Complex

Cytogenetic and molecular studies of a familial paracentric inversion of Y chromosome present in a patient with ambiguous genitalia

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