Protein kinase A and two phosphatases are components of the inositol 1,4,5-trisphosphate receptor macromolecular signaling complex

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed intracellular calcium (Ca(2+)) release channel on the endoplasmic reticulum. IP3Rs play key roles in controlling Ca(2+) signals that activate numerous cellular functions including T cell activation, neurotransmitter release, oocyte fertilization and apoptosis. There are three forms of IP3R, all of which are ligand-gated channels activated by the second messenger inositol 1,4,5-trisphosphate. Channel function is modulated via cross-talk with other signaling pathways including those mediated by kinases and phosphatases. In particular IP3Rs are known to be regulated by cAMP-dependent protein kinase (PKA) phosphorylation. In the present study we show that PKA and the protein phosphatases PP1 and PP2A are components of the IP3R1 macromolecular signaling complex. PKA phosphorylation of IP3R1 increases channel activity in planar lipid bilayers. These studies indicate that regulation of IP3R1 function via PKA phosphorylation involves components of a macromolecular signaling complex.     

Selective phosphorylation of the IP3R-I in vivo by cGMP-dependent protein kinase in smooth muscle

This study examined the expression of inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) types and PKG isoforms in isolated gastric smooth muscle cells and determined the ability of PKG and PKA to phosphorylate IP(3)Rs and inhibit IP(3)-dependent Ca(2+) release, which mediates the initial phase of agonist-induced contraction. PKG-Ialpha and PKG-Ibeta were expressed in gastric smooth muscle cells, together with IP(3)-R-associated cG-kinase substrate, a protein that couples PKG-Ibeta to IP(3)R-I. IP(3)R-I and IP(3)R-III were also expressed, but only IP(3)R-I was phosphorylated by PKA and PKG in vitro and exclusively by PKG in vivo. Sequential phosphorylation by PKA and by PKG-Ialpha in vitro showed that PKA phosphorylated the same site as PKG (presumably S(1755)) and an additional PKA-specific site (S(1589)). In intact muscle cells, agents that activated PKG or both PKG and PKA induced IP(3)R-I phosphorylation that was reversed by the PKG inhibitor (8R,9S,11s)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,1H,-2,7b,11a-trizadizo-benzo9(a,g)cycloocta(c,d,e)-trinden-1-one. Agents that activated PKA induced IP(3)R-I phosphorylation in permeabilized but not intact muscle cells, implying that PKA does not gain access to IP(3)R-I in intact muscle cells. The pattern of IP(3)R-I phosphorylation in vivo and in vitro was more consistent with phosphorylation by PKG-Ialpha. Phosphorylation of IP(3)R-I in microsomes by PKG, PKA, or a combination of PKG and PKA inhibited IP(3)-induced Ca(2+) release to the same extent, implying that inhibition was mediated by phosphorylation of the PKG-specific site. We conclude that IP(3)R-I is selectively phosphorylated by PKG-I in intact smooth muscle resulting in inhibition of IP(3)-dependent Ca(2+) release.     

Phosphorylation of the inositol 1,4,5-trisphosphate receptor by cyclic nucleotide-dependent kinases in vitro and in rat cerebellar slices in situ

We have examined cyclic nucleotide-regulated phosphorylation of the neuronal type I inositol 1,4,5-trisphosphate (IP3) receptor immunopurified from rat cerebellar membranes in vitro and in rat cerebellar slices in situ. The isolated IP3 receptor protein was phosphorylated by both cAMP- and cGMP-dependent protein kinases on two distinct sites as determined by thermolytic phosphopeptide mapping, phosphopeptide 1, representing Ser-1589, and phosphopeptide 2, representing Ser-1756 in the rat protein (Ferris, C. D., Cameron, A. M., Bredt, D. S., Huganir, R. L., and Snyder, S. H. (1991) Biochem. Biophys. Res. Commun. 175, 192-198). Phosphopeptide maps show that cAMP-dependent protein kinase (PKA) labeled both sites with the same time course and same stoichiometry, whereas cGMP-dependent protein kinase (PKG) phosphorylated Ser-1756 with a higher velocity and a higher stoichiometry than Ser-1589. Synthetic decapeptides corresponding to the two phosphorylation sites (peptide 1, AARRDSVLAA (Ser-1589), and peptide 2, SGRRESLTSF (Ser-1756)) were used to determine kinetic constants for the phosphorylation by PKG and PKA, and the catalytic efficiencies were in agreement with the results obtained by in vitro phosphorylation of the intact protein. In cerebellar slices prelabeled with [32P]orthophosphate, activation of endogenous kinases by incubation in the presence of cAMP/cGMP analogues and specific inhibitors of PKG and PKA induced in both cases a 3-fold increase in phosphorylation of the IP3 receptor. Thermolytic phosphopeptide mapping of in situ labeled IP3 receptor by PKA showed labeling on the same sites (Ser-1589 and Ser-1756) as in vitro. In contrast to the findings in vitro, PKG preferentially phosphorylated Ser-1589 in situ. Because both PKG and the IP3 receptor are specifically enriched in cerebellar Purkinje cells, PKG may be an important IP3 receptor regulator in vivo.     

Modes of inhibitory action of protein kinase C in the chemotactic peptide-induced formation of inositol phosphates in differentiated human leukemic (HL-60) cells

Using the [3H]inositol-labeled plasma membranes isolated from the differentiated human leukemic (HL-60) cells, the mode of inhibitory action of the Ca2+/phospholipid-dependent enzyme protein kinase C in the chemotactic peptide, fMet-Leu-Phe (fMLP)-induced, phospholipase C-mediated hydrolysis of phosphoinositides was investigated. In this cell-free membrane system, fMLP in the presence of GTP plus Ca2+, GTP in the presence of Ca2+, or Ca2+ alone could induce the formation of inositol bis- and trisphosphate (IP2 and IP3, respectively). When the intact cells were pre-treated with 12-O-tetradecanoylphorbol-13-acetate, the fMLP- and GTP-induced formation of IP2 and IP3 was markedly reduced but the Ca2+-induced reactions were not reduced in the isolated membranes. This result suggests that protein kinase C impairs the coupling of the GTP-binding protein to the phospholipase C. In another experiment, preincubation of the isolated membranes with pure rat brain protein kinase C inhibited the fMLP-induced formation of IP2, but did not inhibit the GTP- or Ca2+-induced reaction. Under the same conditions, protein kinase C did not inhibit the fMLP-, GTP-, or Ca2+-induced formation of IP3. This result suggests that protein kinase C impairs additionally the coupling of the fMLP receptor to the GTP-binding protein leading to the formation of IP2. The reason for the failure of protein kinase C to inhibit the fMLP-induced formation of IP3 in the cell-free membrane system is unknown, but several possible mechanisms are discussed.     

Activation of platelet-activating factor receptor-coupled G alpha q leads to stimulation of Src and focal adhesion kinase via two separate pathways in human umbilical vein endothelial cells

Platelet-activating factor (PAF), a phospholipid second messenger, has diverse physiological functions, including responses in differentiated endothelial cells to external stimuli. We used human umbilical vein endothelial cells (HUVECs) as a model system. We show that PAF activated pertussis toxin-insensitive G alpha(q) protein upon binding to its seven transmembrane receptor. Elevated cAMP levels were observed via activation of adenylate cyclase, which activated protein kinase A (PKA) and was attenuated by a PAF receptor antagonist, blocking downstream activity. Phosphorylation of Src by PAF required G alpha(q) protein and adenylate cyclase activation; there was an absolute requirement of PKA for PAF-induced Src phosphorylation. Immediate (1 min) PAF-induced STAT-3 phosphorylation required the activation of G alpha(q) protein, adenylate cyclase, and PKA, and was independent of these intermediates at delayed (30 min) and prolonged (60 min) PAF exposure. PAF activated PLC beta 3 through its G alpha(q) protein-coupled receptor, whereas activation of phospholipase C gamma 1 (PLC gamma 1) by PAF was independent of G proteins but required the involvement of Src at prolonged PAF exposure (60 min). We demonstrate for the first time in vascular endothelial cells: (i) the involvement of signaling intermediates in the PAF-PAF receptor system in the induction of TIMP2 and MT1-MMP expression, resulting in the coordinated proteolytic activation of MMP2, and (ii) a receptor-mediated signal transduction cascade for the tyrosine phosphorylation of FAK by PAF. PAF exposure induced binding of p130(Cas), Src, SHC, and paxillin to FAK. Clearly, PAF-mediated signaling in differentiated endothelial cells is critical to endothelial cell functions, including cell migration and proteolytic activation of MMP2.     

Domain-domain interaction of P-Rex1 is essential for the activation and inhibition by G protein betagamma subunits and PKA

PtdIns(3, 4, 5)P(3)-dependent Rac exchanger (P-Rex) 1 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rac. P-Rex1 is activated by G protein betagamma subunits (Gbetagamma), and the Gbetagamma-induced activation is inhibited by cAMP-dependent protein kinase A (PKA). However, the details of regulatory mechanism of P-Rex1 remain to be clarified. In the present study, we investigated the mechanism of activation and inhibition of P-Rex1 using various truncated and alanine-substituted mutants and found that the domain-domain interaction of P-Rex1 is important for Gbetagamma-induced activation and PKA-induced inhibition. Immunoprecipitation analysis showed that the second Disheveled/EGL-10/Pleckstrin (DEP) and first PSD-95/Dlg/ZO-1 (PDZ) domains of P-Rex1 associate with the inositol polyphosphate-4-phosphatase (IP4P) domain. Carboxyl-terminal truncation on the IP4P domain or mutations in the protein-binding pocket of the first PDZ domain abolished the association. Analysis of in vitro guanine nucleotide exchange assay, PAK1/2 phosphorylation, and Rac-specific actin reorganization revealed that Gbetagamma could activate a complex of the P-Rex1 mutant lacking the IP4P domain and the isolated IP4P domain as well as full-length P-Rex1. Moreover, PKA phosphorylation prevented the domain-domain interaction and Gbetagamma-binding. These results provide a new insight into the regulation of other Rho-family GEFs and cell responses induced by the heterotrimeric G protein.     

P2-purinergic receptors activate a guanine nucleotide-dependent phospholipase C in membranes from HL-60 cells

We have previously determined that human neutrophils and monocytes, as well as neutrophil/monocyte progenitor cells, express a subtype of P2-purinergic receptors (for ATP) which activate the inositol phospholipid signalling system. In the present study, membranes prepared from HL-60 promyelocytic leukemia cells were used to examine the mechanism by which these ATP receptors activate phosphatidylinositol-specific phospholipase C (PI-PLC) under defined in vitro conditions. Micromolar concentrations of the receptor agonists ATP, UTP, and ATP gamma S stimulated the GTP-dependent formation of inositol bisphosphate (IP2) and inositol trisphosphate (IP3) in washed membranes prepared from undifferentiated HL-60 cells prelabeled with [3H]inositol. The stimulatory effects of these nucleotides on PI-PLC appeared to be mediated through a GTP binding protein since minimal inositol polyphosphate accumulation was observed in the absence of guanine nucleotides. The increased inositol polyphosphate formation triggered by these nucleotide receptor agonists did not result from inhibition of GTP breakdown. Neither was it a consequence of increased [3H]polyphosphatidylinositol levels resulting from enhanced activity of membrane-associated PI- or PIP-kinases. Instead, the stimulated phospholipase activity was apparently receptor-mediated. The rank order of potency observed in these in vitro membrane assays (ATP = UTP greater than ATP gamma S much greater than TTP greater than CTP much greater than beta, gamma-CH-ATP) was similar to that observed with intact HL-60 cells. This order of potency appears to distinguish the P2-purinergic receptors expressed by human phagocytic leukocytes from the P2 gamma-purinergic receptors which activate PI-PLC in turkey erythrocyte membranes.     

Adenylate cyclase of myometrium and its regulation by cAMP-dependent protein kinase

The purpose of this study was to examine activity of adenylate cyclase (AC) and its cAMP-dependent phosphorylation by cAMP-dependent protein kinase (PKA) in the membrane of rabbit myometrium. Isoproterenol (IP) significantly increased AC activity of nonpregnant rabbits myometrium plasma membranes. However, during pregnancy AC of myometrium plasma membranes did not respond to IP. Phosphorylation of the myometrium membrane by PKA was followed by significantly decreased response of AC to IP.     

Protein kinase A-mediated phosphorylation of serine 357 of the mouse prostacyclin receptor regulates its coupling to G(s)-, to G(i)-, and to G(q)-coupled effector signaling.

The prostacyclin receptor (IP) is primarily coupled to G alpha(s)-dependent activation of adenylyl cyclase; however, a number of studies indicate that the IP may couple to other secondary effector systems perhaps in a species-specific manner. In the current study, we investigated the specificity of G protein:effector coupling by the mouse (m) IP overexpressed in human embryonic kidney 293 cells and endogenously expressed in murine erythroleukemia cells. The mIP exhibited efficient G alpha(s) coupling and concentration-dependent increases in cAMP generation in response to the IP agonist cicaprost; however, mIP also coupled to G alpha(i) decreasing the levels of cAMP in forskolin-treated cells. mIP coupling to G alpha(i) was pertussis toxin-sensitive and was dependent on protein kinase (PK) A activation status. In addition, the mIP coupled to phospholipase C (PLC) activation in a pertussis toxin-insensitive, G alpha(i)-, G beta gamma-, and PKC-independent but in a G alpha(q)- and PKA-dependent manner. Whole cell phosphorylation assays demonstrated that the mIP undergoes cicaprost-induced PKA phosphorylation. mIP(S357A), a site-directed mutant of mIP, efficiently coupled to G alpha(s) but failed to couple to G alpha(i) or to efficiently couple to G alpha(q):PLC. Moreover, mIP(S357A) did not undergo cicaprost-induced phosphorylation confirming that Ser(357) is the target residue for PKA-dependent phosphorylation. Finally, co-precipitation experiments permitted the detection of G alpha(s), G alpha(i), and G alpha(q) in the immunoprecipitates of mIP, whereas only G alpha(s) was co-precipitated with mIP(S357A) indicating that Ser(357) of mIP is essential for G alpha(i) and G alpha(q) interaction. Moreover, inhibition of PKA blocked co-precipitation of mIP with G alpha(i) or G alpha(q). Taken together our data indicate that the mIP, in addition to coupling to G alpha(s), couples to G alpha(i) and G alpha(q); however, G alpha(i) and G alpha(q) coupling is dependent on initial cicaprost-induced mIP:G alpha(s) coupling and phosphorylation of mIP by cAMP-dependent PKA where Ser(357) was identified as the target residue for PKA phosphorylation.     

Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)-regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3β, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3β and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3β increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3β knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3β interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3β interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.     

Phosphorylation of claudin-3 at threonine 192 by cAMP-dependent protein kinase regulates tight junction barrier function in ovarian cancer cells

Claudins are integral membrane proteins essential in the formation and function of tight junctions (TJs). Disruption of TJs, which have essential roles in cell permeability and polarity, is thought to contribute to epithelial tumorigenesis. Claudin-3 and -4 are frequently overexpressed in ovarian cancer, but the molecular pathways involved in the regulation of these proteins are unclear. Interestingly, several studies have demonstrated a role for phosphorylation in the regulation of TJ complexes, although evidence for claudin phosphorylation is scarce. Here, we showed that claudin-3 and -4 can be phosphorylated in ovarian cancer cells. In vitro phosphorylation assays using glutathione S-transferase fusion constructs demonstrated that the C terminus of claudin-3 is an excellent substrate for cAMP-dependent protein kinase (PKA). Using site-directed mutagenesis, we identified a PKA phosphorylation site at amino acid 192 in the C terminus of claudin-3. Overexpression of the protein containing a T192D mutation, mimicking the phosphorylated state, resulted in a decrease in TJ strength in ovarian cancer cell line OVCA433. Our results suggest that claudin-3 phosphorylation by PKA, a kinase frequently activated in ovarian cancer, may provide a mechanism for the disruption of TJs in this cancer. In addition, our findings may have general implications for the regulation of TJs in normal epithelial cells.     

血管内皮屏障功能调节的研究进展

血管内皮屏障功能的调节机制相当复杂。α－凝血酶等炎性介质引起内皮通透生增高的机制可能是通过Ｇ蛋白激活磷脂酶,介导三磷酸肌醇等二信使产生,并进一步激活蛋白激酶Ｃ和肌球蛋白轻链激酶,最终引起肌球蛋白轻链磷酸化,从而导致内皮细胞Ｆ－肌动蛋白骨架重排,中心张力增加,细胞间裂隙形成,内皮细胞通透性发生改变。     

Two waves of the nuclear phospholipase C activity in serum-stimulated HL-60 cells during G(1) phase of the cell cycle

Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cell cycle progression. We have previously demonstrated two peaks of an increase in the nuclear PI-PLC activities in nocodazole-synchronized HL-60 cells. In this study, the activity of nuclear PI-PLC was investigated in serum-stimulated HL-60 cells. In serum-starved HL-60 cells, two peaks of the activity of nuclear PI-PLC were detected at 30 min and 11 h after the re-addition of serum with no parallel increase in PLC activity in cytosol, postnuclear membranes or total cell lysates. An increase in the serine phosphorylation of b splicing variant of PI-PLCbeta(1) was detected with no change in the amount of PI-PLCbeta(1b) in nuclei isolated at 30 min and 11 h after the addition of serum. PI-PLC inhibitor ET-18-OCH(3) and MEK inhibitor PD 98059 completely abolished serum-mediated increase at both time-points. The addition of inhibitors either immediately or 6 h after the addition of serum had inhibitory effects on the number of cells entering S phase. These results demonstrate that two waves of nuclear PI-PLCbeta(1b) activity occur in serum-stimulated cells during G(1) phase of the cell cycle and that the later increase in the PLC activity is equally important for the progression into the S phase.     

G(q)-dependent signalling by the lysophosphatidic acid receptor LPA(3) in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA

The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA(3) receptors, but not LPA(1) and LPA(2), receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the G(alphaq), but not the G(alphai), minigene. Initial contraction and MLC(20) (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-beta (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC(20) and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IkappaBalpha were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr(696) was masked by phosphorylation of MYPT1 at Ser(695) by PKA derived from IkappaB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-beta and MLCK and phosphorylation of MLC(20), and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC(20). Although Rho kinase was activated, phosphorylation of MYPT1 at Thr(696) by Rho kinase was masked by phosphorylation of MYPT1 at Ser(695) via cAMP-independent PKA derived from the NF-kappaB pathway.     

Phosphorylation of lamin B at the nuclear membrane by activated protein kinase C

Both bryostatin 1 and 4 beta-phorbol 12,13-dibutyrate (PBt2) activate Ca2+- and phospholipid-dependent protein kinase (protein kinase C) at the plasma membrane in HL-60 cells (Kraft, A. S., Baker, V. V., and May, W. S. (1987) Oncogene 1, 91-100). However, whereas PBt2 causes HL-60 cells to cease dividing and differentiate, bryostatin 1 antagonizes this effect and allows cells to continue proliferating. To test whether these divergent effects could be due to the differential activation of protein kinase C at the nuclear level, the phosphorylation of nuclear envelope polypeptides was evaluated in cells treated with either bryostatin 1 or PBt2. Bryostatin 1, either alone or in combination with PBt2, but not PBt2 alone, mediates rapid and specific phosphorylation of several nuclear envelope polypeptides. A major target for bryostatin-induced phosphorylation is the major nuclear envelope polypeptide lamin B (Mr = 67,000, pI 6.0). In vitro studies combining purified protein kinase C and HL-60 cell nuclear envelopes demonstrate that bryostatin activates protein kinase C to phosphorylate lamin B, whereas PBt2 does so only weakly, suggesting selective activation of this enzyme toward this substrate. Comparative phosphopeptide and phosphoamino acid analyses demonstrate that bryostatin induces phosphorylation of identical serine sites on lamin B both in whole cells and in vitro. Treatment of whole cells with bryostatin, but not PBt2, leads to specific translocation of activated protein kinase C to the nuclear envelope. Since phosphorylation of lamin B is known to be involved in nuclear lamina depolymerization at the time of mitosis, it is possible that bryostatin-activated protein kinase C activity is involved in this process. Finally, specific activation of protein kinase C at the nuclear membrane could explain, at least in part, the divergent effects of bryostatin 1 and PBt2 on HL-60 cell growth.     

The role of nucleoside-diphosphate kinase reactions in G protein activation of NADPH oxidase by guanine and adenine nucleotides

NADPH-oxidase-catalyzed superoxide (O2-) formation in membranes of HL-60 leukemic cells was activated by arachidonic acid in the presence of Mg2+ and HL-60 cytosol. The GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] and guanosine 5'-[beta,gamma-imido]triphosphate, being potent activators of guanine-nucleotide-binding proteins (G proteins), stimulated O2- formation up to 3.5-fold. The adenine analogue of GTP[gamma S], adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), which can serve as donor of thiophosphoryl groups in kinase-mediated reactions, stimulated O2- formation up to 2.5-fold, whereas the non-phosphorylating adenosine 5'-[beta,gamma-imido]triphosphate was inactive. The effect of ATP[gamma S] was half-maximal at a concentration of 2 microM, was observed in the absence of added GDP and occurred with a lag period two times longer than the one with GTP[gamma S]. HL-60 membranes exhibited nucleoside-diphosphate kinase activity, catalyzing the thiophosphorylation of GDP to GTP[gamma S] by ATP[gamma S]. GTP[gamma S] formation was half-maximal at a concentration of 3-4 microM ATP[gamma S] and was suppressed by removal of GDP by creatine kinase/creatine phosphate (CK/CP). The stimulatory effect of ATP[gamma S] on O2- formation was abolished by the nucleoside-diphosphate kinase inhibitor UDP. Mg2+ chelation with EDTA and removal of endogenous GDP by CK/CP abolished NADPH oxidase activation by ATP[gamma S] and considerably diminished stimulation by GTP[gamma S]. GTP[gamma S] also served as a thiophosphoryl group donor to GDP, with an even higher efficiency than ATP[gamma S]. Transthiophosphorylation of GDP to GTP[gamma S] was only partially inhibited by CK/CP. Our results suggest that NADPH oxidase is regulated by a G protein, which may be activated either by exchange of bound GDP by guanosine triphosphate or by thiophosphoryl group transfer to endogenous GDP by nucleoside-diphosphate kinase.     

Disparate effects of activators of protein kinase C on HL-60 promyelocytic leukemia cell differentiation

In previously published studies (Kreutter, D., Caldwell, A. B., and Morin, M. J. (1985) J. Biol. Chem. 260, 5979-5984), we demonstrated that the activation of the calcium- and phospholipid-dependent protein kinase C by phorbol esters was dissociable from the induction of monocytic differentiation by these agents in HL-60 promyelocytic leukemia cells. We have now compared the effects of two related diterpenes (mezerein and 12-O-tetradecanoylphorbol-13-acetate) and two cell-permeable diacylglycerols (1-oleoyl-2-acetoylglycerol and 1,2-dioctanoylglycerol) on the induction of differentiation in HL-60 cells. Each of these agents activated protein kinase C in vitro and stimulated the phosphorylation of a number of identical proteins in intact HL-60 cells. Exposure to either of the diterpenes at nanomolar concentrations resulted in an inhibition of cell growth and the induction of qualitatively distinct types of monocytic maturation in HL-60 cells. Conversely, neither of the two diacylglycerols was found to be a potent or efficacious inducer of differentiation, as measured by increases in cell adhesion, nonspecific esterase activity, or phagocytosis, even at growth-inhibitory concentrations. However, concurrent exposure of HL-60 cells to both 1,2-dioctanoylglycerol and the calcium ionophore A23187, at concentrations which were without maturational activity when used separately, resulted in measurable increases in both protein phosphorylation and in the fraction of cells expressing a differentiated phenotype. Taken together, these results suggest that specific biochemical effects associated with 12-O-tetradecanoylphorbol-13-acetate, in addition to the activation of protein kinase C, may be important determinants for the induction of leukemia cell differentiation.     

Failure of 1-oleoyl-2-acetylglycerol to mimic the cell-differentiating action of 12-O-tetradecanoylphorbol 13-acetate in HL-60 cells

Treatment of human promyelocytic leukemia cells (HL-60 cells) with 12-O-tetradecanoylphorbol 13-acetate (TPA) results in terminal differentiation of the cells to macrophage-like cells. Treatment of the cells with TPA induced marked enhancement of the phosphorylation of 28- and 67-kDa proteins and a decrease in that of a 75-kDa protein. When the cells were treated with diacylglycerol, i.e. 50 micrograms/ml 1-oleoyl-2-acetylglycerol (OAG), similar changes in the phosphorylation of 28-, 67-, and 75-kDa proteins were likewise observed, indicating that OAG actually stimulates protein kinase C in intact HL-60 cells. OAG (1-100 micrograms/ml), which we used, activated partially purified mouse brain protein kinase C in a concentration-dependent manner. Treatment of HL-60 cells with 10 nM TPA for 48 h caused an increase by about 8-fold in cellular acid phosphatase activity. Although a significant increase in acid phosphatase activity was induced by OAG, the effect was scant compared to that of TPA (less than 7% that of TPA). After 48-h exposure to 10 nM TPA, about 95% of the HL-60 cells adhered to culture dishes. On the contrary, treatment of the cells either with OAG (2-100 micrograms/ml) or phospholipase C failed to induce HL-60 cell adhesion. Ca2+ ionophore A23187 failed to act synergistically with OAG. In addition, hourly or bi-hourly cumulative addition of OAG for 24 h also proved ineffective to induce HL-60 cell adhesion. Our present results do not imply that protein kinase C activation is nonessential for TPA-induced HL-60 cell differentiation, but do demonstrate that protein kinase C activation is not the sole event sufficient to induce HL-60 cell differentiation by means of this agent.