Identification of Essential Residues for the Catalytic Function of 85-kDa Cytosolic Phospholipase A2

Pickard, Richard T.; Chiou, X. Grace; Strifler, Beth A.; DeFelippis, Michael R.; Hyslop, Paul A.; Tebbe, Ann Louise; Yee, Ying K.; Reynolds, Laure J.; Dennis, Edward A.; Kramer, Ruth M.; Sharp, John

doi:10.1074/jbc.271.32.19225

Cited by 104 publications

(87 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The second block contains the Asp of the catalytic dyad (Asp-344 in ExoU). Mutagenesis has confirmed the functional importance of the Ser and Asp in cPLA 2 s and iPLA 2 s (33)(34)(35)(36)(37)(38)(39).…”

Section: Resultsmentioning

confidence: 99%

In Vivo Phospholipase Activity of the Pseudomonas aeruginosa Cytotoxin ExoU and Protection of Mammalian Cells with Phospholipase A2 Inhibitors

Phillips

Six

Dennis

et al. 2003

Journal of Biological Chemistry

Self Cite

165

202

View full text Add to dashboard Cite

A number of clinical isolates of Pseudomonas aeruginosa are cytotoxic to mammalian cells due to the action of the 74-kDa protein ExoU, which is secreted into host cells by the type III secretion system and whose function is unknown. Here we report that the swift and profound cytotoxicity induced by purified ExoU or by an ExoUexpressing strain of P. aeruginosa is blocked by various inhibitors of cytosolic (cPLA 2 ) and Ca 2؉ -independent (iPLA 2 ) phospholipase A 2 enzymes. In contrast, no cytoprotection is offered by inhibitors of secreted phospholipase A 2 enzymes or by a number of inhibitors of signal transduction pathways. This suggests that phospholipase A 2 inhibitors may represent a novel mode of treatment for acute P. aeruginosa infections. We find that 300 -600 molecules of ExoU/cell are required to achieve half-maximal cell killing and that ExoU localizes to the host cell plasma membrane in punctate fashion. We also show that ExoU interacts in vitro with an inhibitor of cPLA 2 and iPLA 2 enzymes and contains a putative serine-aspartate catalytic dyad homologous to those found in cPLA 2 and iPLA 2 enzymes. Mutation of either the serine or the aspartate renders ExoU non-cytotoxic. Although no phospholipase or esterase activity is detected in vitro, significant phospholipase activity is detected in vivo, suggesting that ExoU requires one or more host cell factors for activation as a membrane-lytic and cytotoxic phospholipase.

show abstract

Section: Resultsmentioning

confidence: 99%

In Vivo Phospholipase Activity of the Pseudomonas aeruginosa Cytotoxin ExoU and Protection of Mammalian Cells with Phospholipase A2 Inhibitors

Phillips

Six

Dennis

et al. 2003

Journal of Biological Chemistry

Self Cite

165

202

View full text Add to dashboard Cite

show abstract

“…7C). The three-dimensional structure of cPLA 2 has been elucidated recently [37] confirming that the amino acids of the active site lie within a large C-terminal domain of the enzyme [6,38]. We could not rule out that the addition of a C-terminal sequence to cPLA2 might have disturbed the conformation of the enzyme resulting in decreased activity.…”

Section: Discussionmentioning

confidence: 99%

N‐terminal and C‐terminal plasma membrane anchoring modulate differently agonist‐induced activation of cytosolic phospholipase A₂

Klapisz

Ziari

Wendum

et al. 1999

European Journal of Biochemistry

View full text Add to dashboard Cite

The 85 kDa cytosolic phospholipase A 2 (cPLA 2 ) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA 2 undergoes calciumdependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA 2 activation, we constructed chimeras of cPLA 2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA 2 ) or the C-terminal targeting signal of K-Ras4B (cPLA 2 -Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the a 2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [ 3 H]arachidonic acid, indicating that constitutive association of cPLA 2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA 2 increased [ 3 H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA 2 -Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA 2 . The lack of stimulation of cPLA 2 -Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.

show abstract

“…Histidines have been implicated in metal ion binding (27) and electrostatic stabilization of intermediates (28). The catalytic importance of arginine has been shown in nucleotide substrate binding (29), and it may form a catalytic triad with serine and asparagine (30). Arginine can also have structural importance, as demonstrated by its role in conformational changes in murine leukemia virus reverse transcriptase (31).…”

mentioning

confidence: 99%

Chemical Modification and Site-directed Mutagenesis of Conserved HXXH and PP-loop Motif Arginines and Histidines in the Murine Bifunctional ATP Sulfurylase/Adenosine 5′-Phosphosulfate Kinase

Deyrup

Singh

Krishnan

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

The sulfurylase domain of the mouse bifunctional enzyme ATP sulfurylase/adenosine 5 -phosphosulfate (APS) kinase contains HXXH and PP-loop motifs. To elucidate the functional importance of these motifs and of conserved arginines and histidines, chemical modification and site-directed mutagenesis studies were performed. Chemical modification of arginines and histidines with phenylglyoxal and diethyl pyrocarbonate, respectively, renders the enzyme inactive in sulfurylase, kinase, and overall assays. Data base searches and sequence comparison of bifunctional ATP sulfurylase/APS kinase and monofunctional ATP sulfurylases shows a limited number of highly conserved arginines and histidines within the sulfurylase domain. Of these conserved residues, His-425, His-428, and Arg-421 are present within or near the HXXH motif whereas His-506, Arg-510, and Arg-522 residues are present in and around the PP-loop. The functional role of these conserved residues was further studied by site-directed mutagenesis. In the HXXH motif, none of the alanine mutants (H425A, H428A, and R421A) had sulfurylase or overall activity, whereas they all exhibited normal kinase activity. A slight improvement in reverse sulfurylase activity (<10% residual activity) and complete restoration of forward sulfurylase was observed with R421K. Mutants designed to probe the PP-loop requirements included H506A, R510A, R522A, R522K, and D523A. Of these, R510A exhibited normal sulfurylase and kinase activity, R522A and R522K showed no sulfurylase activity, and H506A had normal sulfurylase activity but produced an effect on kinase activity (<10% residual activity). The single aspartate, D523A, which is part of the highly conserved GRD sequence of the PP-loop, affected both sulfurylase and kinase activity. This mutational analysis indicates that the HXXH motif plays a role only in the sulfurylase activity, whereas the PP-loop is involved in both sulfurylase and kinase activities. Residues specific for sulfurylase activity have also been distinguished from those involved in kinase activity.Recently there has been increased interest in the sulfateactivating bifunctional enzyme, ATP sulfurylase/APS 1 kinase (PAPS synthetase). With the recent cloning of two isoforms of sulfurylase/kinase (SK) from both mouse, MSK1 (1) and MSK2 (2), and human, HSK1 (3, 4) and HSK2 (3), increased efforts are directed toward understanding the structure-function relationship of the bifunctional enzyme. This enzyme was initially identified by Sugahara and Schwartz (5-7) as the site of the brachymorphic defect in mice. The enzyme was subsequently purified to homogeneity and complete kinetic, functional, and structural analyses of the two-step pathway revealed that both activities reside on a single bifunctional protein that uses a channeling mechanism to efficiently produce PAPS (8 -12). We have also reported the cloning of two mouse bifunctional enzyme isoforms, MSK1 (1) and MSK2 (2), and have shown that a point mutation in the kinase portion of MSK2 renders the enzyme inactive, causing ...

show abstract

Identification of Essential Residues for the Catalytic Function of 85-kDa Cytosolic Phospholipase A2

Cited by 104 publications

References 53 publications

In Vivo Phospholipase Activity of the Pseudomonas aeruginosa Cytotoxin ExoU and Protection of Mammalian Cells with Phospholipase A2 Inhibitors

In Vivo Phospholipase Activity of the Pseudomonas aeruginosa Cytotoxin ExoU and Protection of Mammalian Cells with Phospholipase A2 Inhibitors

N‐terminal and C‐terminal plasma membrane anchoring modulate differently agonist‐induced activation of cytosolic phospholipase A₂

Chemical Modification and Site-directed Mutagenesis of Conserved HXXH and PP-loop Motif Arginines and Histidines in the Murine Bifunctional ATP Sulfurylase/Adenosine 5′-Phosphosulfate Kinase

Contact Info

Product

Resources

About

Identification of Essential Residues for the Catalytic Function of 85-kDa Cytosolic Phospholipase A2

Cited by 104 publications

References 53 publications

In Vivo Phospholipase Activity of the Pseudomonas aeruginosa Cytotoxin ExoU and Protection of Mammalian Cells with Phospholipase A2 Inhibitors

In Vivo Phospholipase Activity of the Pseudomonas aeruginosa Cytotoxin ExoU and Protection of Mammalian Cells with Phospholipase A2 Inhibitors

N‐terminal and C‐terminal plasma membrane anchoring modulate differently agonist‐induced activation of cytosolic phospholipase A2

Chemical Modification and Site-directed Mutagenesis of Conserved HXXH and PP-loop Motif Arginines and Histidines in the Murine Bifunctional ATP Sulfurylase/Adenosine 5′-Phosphosulfate Kinase

Contact Info

Product

Resources

About

N‐terminal and C‐terminal plasma membrane anchoring modulate differently agonist‐induced activation of cytosolic phospholipase A₂