Effects of Protein Kinase CK2, Extracellular Signal-regulated Kinase 2, and Protein Phosphatase 2A on a Phosphatidic Acid-preferring Phospholipase A1

Abstract

A soluble, phosphatidic acid-preferring phospholipase A1, expressed in mature bovine testes but not in newborn calf testes, may contribute to the formation or function of sperm. Here we incubated a recombinant preparation of the phospholipase in vitro with several enzymes including protein kinase CK2 (CK2), extracellular signal-regulated kinase 2 (ERK2), and protein phosphatase 2A (PP2A) to identify effects that might be of regulatory importance in vivo. Major findings were that 1) CK2 phosphorylated the phospholipase on serines 93, 105, and 716; 2) ERK2 phosphorylated the enzyme on serine 730; 3) there was cross-antagonism between the reactions that phosphorylated serines 716 and 730; 4) PP2A selectively hydrolyzed phosphate groups that were esterified to serines 716 and 730; 5) CK2␣ formed a stable, MgATP/MgGTP-dependent complex with the phospholipase by a novel mechanism; and 6) the complex showed reduced phospholipase activity and resembled a complex identified in homogenates of macaque testis. These results provide the first available information about the effects of reactions of phosphorylation and dephosphorylation on the behavior of the phospholipase, shed light on properties of CK2␣ that may be required for the formation of complexes with its substrates, and raise the possibility that a complex containing CK2␣ and the phospholipase may play a special biological role in the testis.Mammalian tissues contain a soluble phospholipase A1 that can catalyze the preferential hydrolysis of PA 1 in assays using mixed micelles (1) or unilamellar vesicles (2). The enzyme has been purified to homogeneity from bovine testes (3) and shown to have a molecular mass of 97.6 kDa, as determined by matrixassisted laser desorption/ionization (4). Its cDNA has been cloned and sequenced and shown to encode an 875-amino acid protein that resembles other phospholipases only in so far as it contains a five-amino acid lipase consensus domain that includes a central serine residue (serine 540) required for catalysis (4). Moreover, analyses of the distribution of the human enzyme's mRNA have provided evidence that this enzyme and one of its splice variants are expressed selectively in human tissues. 2 However, the enzyme's biological role remains to be determined, and little is known about the regulation of its activity inside cells.The aim of the present investigation was to explore the possibility that protein kinases and phosphatases might affect the behavior of the first identified (bovine) splice variant of the enzyme, which we now call PA-PLA1␣. We expressed an affinity-tagged, recombinant form of this enzyme in Sf9 cells, purified it, and examined the ability of several protein kinases and phosphatases to phosphorylate or dephosphorylate it in vitro. But only CK2 and ERK2 phosphorylated the phospholipase with significant stoichiometry, and only PP2A could catalyze the hydrolysis of the phosphate esters. We used mass spectrometry to identify the amino acids that were phosphorylated or dephosphorylated and used several other app...

p125 Is Localized in Endoplasmic Reticulum Exit Sites and Involved in Their Organization

“…Global localization analysis has suggested that the YOR022c protein is localized in mitochondria (46). These findings combined with our results and the results of others (24,25,47, and this study) suggest that PA-PLA 1 family proteins appear to be localized in diverse subcellular compartments and may be involved in various biological processes.…”

Progesterone Regulates Granulosa Cell Viability Through a Protein Kinase G-Dependent Mechanism That May Involve 14-3-3σ1

“…The amino acids that are preceded by a p are the amino acids that were phosphorylated. The details regarding the methods used to reveal the sequence and the phosphorylation sites are found in the papers published by Han et al [29] and Han et al [30], respectively. though the 8-br-cAMP and P4 have a similar impact on cell viability, treatment with P4 does not increase PKA activity, as assessed by the phosphorylation of cAMP response element binding protein (CREB) [32].…”

“…To determine which site or sites were phosphorylated, the 14-3-3 spot was dissected from three gels obtained from P4-treated lysates and the spots pooled into a single sample. This sample was then analyzed for phosphorylated amino acids as outlined by Han et al [30]. This work was done at the Proteomics and Biological Mass Spectrometry Core Facility (University of Connecticut Health Center, Farmington, CT).…”

Molecular features of adult mouse small intestinal epithelial progenitors

“…36) CkII substrates are dephosphorylated by Pp2A (37). In the case of c-myc, this dephosphorylation promotes degradation by the ubiquitin͞proteasomal pathway (37). Erk substrates are also dephosphorylated by Pp2A (37).…”

“…SiEP express Ppp2cb mRNA (encodes the ␤ isoform of the catalytic subunit of the protein phosphatase 2A complex; ref. 36) CkII substrates are dephosphorylated by Pp2A (37). In the case of c-myc, this dephosphorylation promotes degradation by the ubiquitin͞proteasomal pathway (37).…”