Interaction of the HIV-1 Intasome with Transportin 3 Protein (TNPO3 or TRN-SR2)

Transportin 3 (TNPO3 or TRN-SR2) has been shown to be an important cellular factor for early steps of lentiviral replication. However, separate studies have implicated distinct mechanisms for TNPO3 either through its interaction with HIV-1 integrase or capsid. Here we have carried out a detailed biophysical characterization of TNPO3 and investigated its interactions with viral proteins. Biophysical analyses including circular dichroism, analytical ultracentrifugation, small-angle x-ray scattering, and homology modeling provide insight into TNPO3 architecture and indicate that it is highly structured and exists in a monomer-dimer equilibrium in solution. In vitro biochemical binding assays argued against meaningful direct interaction between TNPO3 and the capsid cores. Instead, TNPO3 effectively bound to the functional intasome but not to naked viral DNA, suggesting that TNPO3 can directly engage the HIV-1 IN tetramer prebound to the cognate DNA. Mass spectrometry-based protein footprinting and site-directed mutagenesis studies have enabled us to map several interacting amino acids in the HIV-1 IN C-terminal domain and the cargo binding domain of TNPO3. Our findings provide important information for future genetic analysis to better understand the role of TNPO3 and its interacting partners for HIV-1 replication.     

Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity

The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.     

Enabling association of the GINS protein tetramer with the mini chromosome maintenance (Mcm)2-7 protein complex by phosphorylated Sld2 protein and single-stranded origin DNA

The Cdc45-Mcm2-7-GINS (CMG) complex is the replication fork helicase in eukaryotes. Synthetic lethal with Dpb11-1 (Sld2) is required for the initiation of DNA replication, and the S phase cyclin-dependent kinase (S-CDK) phosphorylates Sld2 in vivo. We purified components of the replication initiation machinery and studied their interactions in vitro. We found that unphosphorylated or CDK-phosphorylated Sld2 binds to the mini chromosome maintenance (Mcm)2-7 complex with similar efficiency. Sld2 interaction with Mcm2-7 blocks the interaction between GINS and Mcm2-7. The interaction between CDK-phosphorylated Sld2 and Mcm2-7 is substantially inhibited by origin single-stranded DNA (ssDNA). Furthermore, origin ssDNA allows GINS to bind to Mcm2-7 in the presence of CDK-phosphorylated Sld2. However, unphosphorylated Sld2 blocks the interaction between GINS and Mcm2-7 even in the presence of origin ssDNA. We identified a mutant of Sld2 that does not bind to DNA. When this mutant is expressed in yeast cells, cell growth is severely inhibited with very slow progression into S phase. We propose a model wherein Sld2 blocks the interaction between GINS and Mcm2-7 in vivo. Once origin ssDNA is extruded from the Mcm2-7 ring and CDK phosphorylates Sld2, the origin ssDNA binds to CDK-phosphorylated Sld2. This event may allow the interaction between GINS and Mcm2-7 in vivo. Thus, CDK phosphorylation of Sld2 may be important to release Sld2 from Mcm2-7, thereby allowing GINS to bind Mcm2-7. Furthermore, origin ssDNA may stimulate the formation of the CMG complex by alleviating inhibitory interactions between Sld2 with Mcm2-7.     

Molecular mechanism of activation of human Cdc7 kinase: bipartite interaction with Dbf4/activator of S phase kinase (ASK) activation subunit stimulates ATP binding and substrate recognition

Cdc7 is a serine/threonine kinase conserved from yeasts to human and is known to play a key role in the regulation of initiation at each replication origin. Its catalytic function is activated via association with the activation subunit Dbf4/activator of S phase kinase (ASK). It is known that two conserved motifs of Dbf4/ASK are involved in binding to Cdc7, and both are required for maximum activation of Cdc7 kinase. Cdc7 kinases possess unique kinase insert sequences (kinase insert I-III) that are inserted at defined locations among the conserved kinase domains. However, precise mechanisms of Cdc7 kinase activation are largely unknown. We have identified two segments on Cdc7, DAM-1 (Dbf4/ASK interacting motif-1; amino acids 448-457 near the N terminus of kinase insert III) and DAM-2 (C-terminal 10-amino acid segment), that interact with motif-M and motif-C of ASK, respectively, and are essential for kinase activation by ASK. The C-terminal 143-amino acid polypeptide (432-574) containing DAM-1 and DAM-2 can interact with Dbf4/ASK. Characterization of the purified ASK-free Cdc7 and Cdc7-ASK complex shows that ATP binding of the Cdc7 catalytic subunit requires Dbf4/ASK. However, the "minimum" Cdc7, lacking the entire kinase insert II and half of kinase insert III, binds to ATP and shows autophosphorylation activity in the absence of ASK. However, ASK is still required for phosphorylation of exogenous substrates by the minimum Cdc7. These results indicate bipartite interaction between Cdc7 and Dbf4/ASK subunits facilitates ATP binding and substrate recognition by the Cdc7 kinase.     

Regulation of protein kinase C-related protein kinase 2 (PRK2) by an intermolecular PRK2-PRK2 interaction mediated by Its N-terminal domain

Protein kinase C-related protein kinases (PRKs) are effectors of the Rho family of small GTPases and play a role in the development of diseases such as prostate cancer and hepatitis C. Here we examined the mechanism underlying the regulation of PRK2 by its N-terminal region. We show that the N-terminal region of PRK2 prevents the interaction with its upstream kinase, the 3-phosphoinositide-dependent kinase 1 (PDK1), which phosphorylates the activation loop of PRK2. We confirm that the N-terminal region directly inhibits the kinase activity of PRK2. However, in contrast to previous models, our data indicate that this inhibition is mediated in trans through an intermolecular PRK2-PRK2 interaction. Our results also suggest that amino acids 487-501, located in the linker region between the N-terminal domains and the catalytic domain, contribute to the PRK2-PRK2 dimer formation. This dimerization is further supported by other N-terminal domains. Additionally, we provide evidence that the region C-terminal to the catalytic domain intramolecularly activates PRK2. Finally, we discovered that the catalytic domain mediates a cross-talk between the inhibitory N-terminal region and the activating C-terminal region. The results presented here describe a novel mechanism of regulation among AGC kinases and offer new insights into potential approaches to pharmacologically regulate PRK2.     

The unstructured C-terminal tail of yeast Dpb11 (human TopBP1) protein is dispensable for DNA replication and the S phase checkpoint but required for the G2/M checkpoint

Budding yeast Dpb11 (human TopBP1, fission yeast Cut5) is an essential protein required for replisome assembly and for the DNA damage checkpoint. Previous studies with the temperature-sensitive dpb11-1 allele, truncated at amino acid 583 of the 764-amino acid protein, have suggested the model that Dpb11 couples DNA replication to the replication checkpoint. However, the dpb11-1 allele shows distinct replication defects even at permissive temperatures. Here, we determine that the 1-600-amino acid domain of DPB11 is both required and sufficient for full replication function of Dpb11 but that this domain is defective for activation of the principal checkpoint kinase Mec1 (human ataxia telangiectasia and Rad3-related) in vitro and in vivo. Remarkably, mutants of DPB11 that leave its replication function intact but abrogate its ability to activate Mec1 are proficient for the replication checkpoint, but they are compromised for the G(2)/M DNA damage checkpoint. These data suggest that replication checkpoint defects may result indirectly from defects in replisome assembly. Two conserved aromatic amino acids in the C terminus of Dpb11 are critical for Mec1 activation in vitro and for the G(2)/M checkpoint in yeast. Together with aromatic motifs identified previously in the Ddc1 subunit of 9-1-1, another activator of Mec1 kinase, they define a consensus structure for Mec1 activation.     

p21-Activated kinase 3 (PAK3) protein regulates synaptic transmission through its interaction with the Nck2/Grb4 protein adaptor

Mutations in the p21-activated kinase 3 gene (pak3) are responsible for nonsyndromic forms of mental retardation. Expression of mutated PAK3 proteins in hippocampal neurons induces abnormal dendritic spine morphology and long term potentiation anomalies, whereas pak3 gene invalidation leads to cognitive impairments. How PAK3 regulates synaptic plasticity is still largely unknown. To better understand how PAK3 affects neuronal synaptic plasticity, we focused on its interaction with the Nck adaptors that play a crucial role in PAK signaling. We report here that PAK3 interacts preferentially with Nck2/Grb4 in brain extracts and in transfected cells. This interaction is independent of PAK3 kinase activity. Selective uncoupling of the Nck2 interactions in acute cortical slices using an interfering peptide leads to a rapid increase in evoked transmission to pyramidal neurons. The P12A mutation in the PAK3 protein strongly decreases the interaction with Nck2 but only slightly with Nck1. In transfected hippocampal cultures, expression of the P12A-mutated protein has no effect on spine morphogenesis or synaptic density. The PAK3-P12A mutant does not affect synaptic transmission, whereas the expression of the wild-type PAK3 protein decreases the amplitude of spontaneous miniature excitatory currents. Altogether, these data show that PAK3 down-regulates synaptic transmission through its interaction with Nck2.     

Arginine methylation of the nuclear poly(a) binding protein weakens the interaction with its nuclear import receptor, transportin

The nuclear poly(A) binding protein, PABPN1, promotes mRNA polyadenylation in the cell nucleus by increasing the processivity of poly(A) polymerase and contributing to poly(A) tail length control. In its C-terminal domain, the protein carries 13 arginine residues that are all asymmetrically dimethylated. The function of this modification in PABPN1 has been unknown. Part of the methylated domain serves as nuclear localization signal, binding the import receptor transportin. Here we report that arginine methylation weakens the affinity of PABPN1 for transportin. Recombinant, unmethylated PABPN1 binds more strongly to transportin than its methylated counterpart from mammalian tissue, and in vitro methylation reduces the affinity. Transportin and RNA compete for binding to PABPN1. Methylation favors RNA binding. Transportin also inhibits in vitro methylation of the protein. Finally, a peptide corresponding to the nuclear localization signal of PABPN1 competes with transportin-dependent nuclear import of the protein in a permeabilized cell assay and does so less efficiently when it is methylated. We hypothesize that transportin binding might delay methylation of PABPN1 until after nuclear import. In the nucleus, arginine methylation may favor the transition of PABPN1 to the competing ligand RNA and serve to reduce the risk of the protein being reexported to the cytoplasm by transportin.     

ICP34.5 protein of herpes simplex virus facilitates the initiation of protein translation by bridging eukaryotic initiation factor 2alpha (eIF2alpha) and protein phosphatase 1

The ICP34.5 protein of herpes simplex virus type 1 is a neurovirulence factor that plays critical roles in viral replication and anti-host responses. One of its functions is to recruit protein phosphatase 1 (PP1) that leads to the dephosphorylation of the α subunit of translation initiation factor eIF2 (eIF2α), which is inactivated by infection-induced phosphorylation. As PP1 is a protein phosphatase with a wide range of substrates, the question remains to be answered how ICP34.5 directs PP1 to specifically dephosphorylate eIF2α. Here we report that ICP34.5 not only binds PP1 but also associates with eIF2α by in vitro and in vivo assays. The binding site of eIF2α is identified at amino acids 233-248 of ICP34.5, which falls in the highly homologous region with human gene growth arrest and DNA damage 34. The interaction between ICP34.5 and eIF2α is independent of the phosphorylation status of eIF2α at serine 51. Deletion mutation of this region results in the failure of dephosphorylation of eIF2α by PP1 and, consequently, interrupts viral protein synthesis and replication. Our data illustrated that the binding between viral protein ICP34.5 and the host eIF2α is crucial for the specific dephosphorylation of eIF2α by PP1. We propose that herpes simplex virus protein ICP34.5 bridges PP1 and eIF2α via their binding motifs and thereby facilitates the protein synthesis and viral replication.     

Methylation of the C-terminal leucine residue of the PP2A catalytic subunit is unnecessary for the catalytic activity and the binding of regulatory subunit (PR55/B)

Protein phosphatase 2A (PP2A) is composed of structural (A), catalytic (C), and regulatory (B) subunits. The catalytic subunit (PP2A(C)) undergoes reversible carboxyl-methylation and -demethylation at its C-terminal leucine residue (Leu309), catalyzed by PP2A-methyltransferase (PMT) and PP2A methylesterase (PME-1), respectively. In this study, we observed that the activity of PP2A was largely unaffected by the addition of PME-1, and that the regulatory subunit (PR55/B) could bind demethylated PP2A(D). Furthermore, to study the precise effect of Leu309 demethylation on PP2A activity, we generated two His(8)-tagged mutant versions of PP2A(C) containing an alanine residue in place of Leu309, and a deletion of Leu309. Both recombinant mutants exhibited phosphatase activity. In addition, we demonstrated that both mutants could constitute a holoenzyme with the regulatory A and B subunits. Our collective results indicate that methylation of Leu309 of PP2A(C) is unnecessary for the PP2A activity and the binding of PR55/B.     

Epstein-Barr nuclear antigen 1 (EBNA1)-dependent recruitment of origin recognition complex (Orc) on oriP of Epstein-Barr virus with purified proteins: stimulation by Cdc6 through its direct interaction with EBNA1

Origin recognition complex (Orc) plays an essential role in directing assembly of prereplicative complex at selective sites on chromosomes. However, Orc from vertebrates is reported to bind to DNA in a sequence-nonspecific manner, and it is still unclear how it selects specific genomic loci and how Cdc6, another conserved AAA(+) factor known to interact with Orc, participates in this process. Replication from oriP, the latent origin of Epstein-Barr virus, provides an excellent model system for the study of initiation on the host chromosomes because it is known to depend on prereplicative complex factors, including Orc and Mcm. Here, we show that Orc is recruited selectively at the essential dyad symmetry element in nuclear extracts in a manner dependent on EBNA1, which specifically binds to dyad symmetry. With purified proteins, EBNA1 can recruit both Cdc6 and Orc independently on a DNA containing EBNA1 binding sites, and Cdc6 facilitates the Orc recruitment by EBNA1. Purified Cdc6 directly binds to EBNA1, whereas association of Orc with EBNA1 requires the presence of the oriP DNA. Nuclease protection assays suggest that Orc associates with DNA segments on both sides adjacent to the EBNA1 binding sites and that this process is stimulated by the presence of Cdc6. Thus, EBNA1 can direct localized assembly of Orc in a process that is facilitated by Cdc6. The possibility of similar modes of recruitment of Orc/Cdc6 at the human chromosomal origins will be discussed.     

A variant of the breast cancer type 2 susceptibility protein (BRC) repeat is essential for the RECQL5 helicase to interact with RAD51 recombinase for genome stabilization

The BRC repeat is a structural motif in the tumor suppressor BRCA2 (breast cancer type 2 susceptibility protein), which promotes homologous recombination (HR) by regulating RAD51 recombinase activity. To date, the BRC repeat has not been observed in other proteins, so that its role in HR is inferred only in the context of BRCA2. Here, we identified a BRC repeat variant, named BRCv, in the RECQL5 helicase, which possesses anti-recombinase activity in vitro and suppresses HR and promotes cellular resistance to camptothecin-induced replication stress in vivo. RECQL5-BRCv interacted with RAD51 through two conserved motifs similar to those in the BRCA2-BRC repeat. Mutations of either motif compromised functions of RECQL5, including association with RAD51, inhibition of RAD51-mediated D-loop formation, suppression of sister chromatid exchange, and resistance to camptothecin-induced replication stress. Potential BRCvs were also found in other HR regulatory proteins, including Srs2 and Sgs1, which possess anti-recombinase activities similar to that of RECQL5. A point mutation in the predicted Srs2-BRCv disrupted the ability of the protein to bind RAD51 and to inhibit D-loop formation. Thus, BRC is a common RAD51 interaction module that can be utilized by different proteins to either promote HR, as in the case of BRCA2, or to suppress HR, as in RECQL5.     

The structural details of the interaction of single-stranded DNA binding protein hSSB2 (NABP1/OBFC2A) with UV-damaged DNA

Single-stranded DNA-binding proteins (SSBs) are required for all known DNA metabolic events such as DNA replication, recombination and repair. While a wealth of structural and functional data is available on the essential human SSB, hSSB1 (NABP2/OBFC2B), the close homolog hSSB2 (NABP1/OBFC2A) remains relatively uncharacterized. Both SSBs possess a well-structured OB (oligonucleotide/oligosaccharide-binding) domain that is able to recognize single-stranded DNA (ssDNA) followed by a flexible carboxyl-tail implicated in the interaction with other proteins. Despite the high sequence similarity of the OB domain, several recent studies have revealed distinct functional differences between hSSB1 and hSSB2. In this study, we show that hSSB2 is able to recognize cyclobutane pyrimidine dimers (CPD) that form in cellular DNA as a consequence of UV damage. Using a combination of biolayer interferometry and NMR, we determine the molecular details of the binding of the OB domain of hSSB2 to CPD-containing ssDNA, confirming the role of four key aromatic residues in hSSB2 (W59, Y78, W82, and Y89) that are also conserved in hSSB1. Our structural data thus demonstrate that ssDNA recognition by the OB fold of hSSB2 is highly similar to hSSB1, indicating that one SSB may be able to replace the other in any initial ssDNA binding event. However, any subsequent recruitment of other repair proteins most likely depends on the divergent carboxyl-tail and as such is likely to be different between hSSB1 and hSSB2.     

The anti-apoptotic effect of IGF-1 on tissue resident stem cells is mediated via PI3-kinase dependent secreted frizzled related protein 2 (Sfrp2) release

Previous studies suggest that IGF-1 may be used as an adjuvant to stem cell transfer in order to improve cell engraftment in ischemic tissue. In the current study, we investigated the effect of IGF-1 on serum deprivation and hypoxia induced stem cell apoptosis and the possible mechanisms involved. Exposure of adipose tissue derived stem cells (ASCs) to serum deprivation and hypoxia resulted in significant apoptosis in ASC which is partially prevented by IGF-1. IGF-1’s anti-apoptotic effect was abolished in ASCs transfected with Sfrp2 siRNA but not by the control siRNA. Using Western blot analysis, we demonstrated that serum deprivation and hypoxia reduced the expression of nuclear β-catenin, which is reversed by IGF-1. IGF-1’s effect on β-catenin expression was abolished by the presence of PI3-kinase inhibitor LY294002 or in ASCs transfected with Sfrp2 siRNA. These results suggest that IGF-1, through the release of the Sfrp2, contributes to cell survival by stabilizing β-catenin.     

Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase‐D (dermonecrotic toxin) from brown spider venom

Brown spiders have world‐wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase‐D , causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose‐dependent and time‐dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP‐phospholipase‐D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin‐treated erythrocytes reacted with annexin‐V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase‐D , which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine‐protease inhibitor) that had no effect on hemolysis. By site‐directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase‐D triggers direct human blood cell hemolysis in a catalytic‐dependent manner. J. Cell. Biochem. 107: 655–666, 2009. © 2009 Wiley‐Liss, Inc.     

phi29 DNA polymerase residue Phe128 of the highly conserved (S/T)Lx(2)h motif is required for a stable and functional interaction with the terminal protein

Bacteriophage phi29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as primer for initiation of DNA replication. By multiple sequence alignments of DNA polymerases from such a family, we have been able to identify two amino acid residues specifically conserved in the protein-priming subgroup of DNA polymerases, a phenylalanine contained in the (S/T)Lx(2)h motif, and a glutamate belonging to the Exo III motif. Here, we have studied the functional role of these residues in reactions that are specific for DNA polymerases that use a protein-primed DNA replication mechanism, by site-directed mutagenesis in the corresponding amino acid residues, Phe128 and Glu161 of phi29 DNA polymerase. Mutations introduced at residue Phe128 severely impaired the protein-primed replication capacity of the polymerase, being the interaction with the terminal protein (TP) moderately (mutant F128A) or severely (mutant F128Y) diminished. As a consequence, very few initiation products were obtained, and essentially no transition products were detected. Interestingly, phi29 DNA polymerase mutant F128Y showed a decreased binding affinity for short template DNA molecules. These results, together with the high degree of conservation of Phe128 residue among protein-primed DNA polymerases, suggest a functional role for this amino acid residue in making contacts with the TP during the first steps of genome replication and with DNA in the further replication steps.     

The activity of aminoacyl-tRNA synthetase-interacting multi-functional protein 1 (AIMP1) on endothelial cells is mediated by the assembly of a cytoskeletal protein complex

AIMP1 was first found as a factor associated with the aminoacyl-tRNA synthetase (ARS) complex. However, it is also secreted and acts on different target cells such as endothelial cells, macrophages, and fibroblasts as an extracellular regulator, respectively, of angiogenesis, inflammatory responses and dermal regeneration. AIMP1 has also been reported to suppress in vivo tumor growth. In this study, we investigated the signaling pathways activated by exogenous AIMP1 in an in vitro endothelial model. AIMP1 decreases EC viability through an α5β1 integrin-dependent mechanism and inhibits cell adhesion, is internalized and shows an asymmetric pattern of distribution and accumulation in cell protrusions. Experiments of affinity purification, pull down, and co-immunoprecipitation showed that AIMP1 interacts with four cytoskeletal proteins (filamin-A, α-tubulin, vinculin, and cingulin). α-Tubulin also gets phosphorylated upon cell treatment with AIMP1 and colocalization between AIMP1 and filamin-A as well as between AIMP1 and cingulin was observed through immunofluorescence assays. In this work, we propose that AIMP1 effect on EC adhesion is mediated by the assembly of a cytoskeletal protein complex on the cytosolic face of the cell membrane which could regulate cellular architecture maintenance and remodeling. Moreover, this activity is able to indirectly influence cell viability.     

The effect of 24R,25-(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C

1alpha,25-(OH)(2)D(3) regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D(3) regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)(2)D(3) and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D(3) metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D(3). Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)(2)D(3). This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D(3) on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(2)D(3)-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R,25-(OH)(2)D(3)-stimulated PKC in RC cells and stimulated basal and 1alpha,25-(OH)(2)D(3)-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,25-(OH)(2)D(3) regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D(3) regulates PKC in RC cells via PLD2.