Rho kinase, myosin-II, and p42/44 MAPK control extracellular matrix-mediated apical bile canalicular lumen morphogenesis in HepG2 cells

The molecular mechanisms that regulate multicellular architecture and the development of extended apical bile canalicular lumens in hepatocytes are poorly understood. Here, we show that hepatic HepG2 cells cultured on glass coverslips first develop intercellular apical lumens typically formed by a pair of cells. Prolonged cell culture results in extensive organizational changes, including cell clustering, multilayering, and apical lumen morphogenesis. The latter includes the development of large acinar structures and subsequent elongated canalicular lumens that span multiple cells. These morphological changes closely resemble the early organizational pattern during development, regeneration, and neoplasia of the liver and are rapidly induced when cells are cultured on predeposited extracellular matrix (ECM). Inhibition of Rho kinase or its target myosin-II ATPase in cells cultured on glass coverslips mimics the morphogenic response to ECM. Consistently, stimulation of Rho kinase and subsequent myosin-II ATPase activity by lipoxygenase-controlled eicosatetranoic acid metabolism inhibits ECM-mediated cell multilayering and apical lumen morphogenesis but not initial apical lumen formation. Furthermore, apical lumen remodeling but not cell multilayering requires basal p42/44 MAPK activity. Together, the data suggest a role for hepatocyte-derived ECM in the spatial organization of hepatocytes and apical lumen morphogenesis and identify Rho kinase, myosin-II, and MAPK as potentially important players in different aspects of bile canalicular lumen morphogenesis.     

Stimulus-induced translocation of the protein kinase A catalytic subunit to the apical membrane in blowfly salivary glands

Secretion in blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT), which activates the InsP₃/Ca²⁺ pathway and the cAMP/protein kinase A (PKA) pathway in the secretory cells. The latter signaling cascade induces the activation of a vacuolar H⁺-ATPase on the apical membrane. Here, we have determined the distribution of PKA by using antibodies against the PKA regulatory subunit-II (PKA-RII) and the PKA catalytic subunit (PKA-C) of Drosophila. PKA is present in high concentrations within the secretory cells. PKA-RII and PKA-C co-distribute in non-stimulated glands, being enriched in the basal portion of the secretory cells. Exposure to 8-CPT-cAMP or 5-HT induces the translocation of PKA-C to the apical membrane, whereas the PKA-RII distribution remains unchanged. The recruitment of PKA-C to the apical membrane corroborates our hypothesis that vacuolar H⁺-ATPase, which is enriched in this membrane domain, is a target protein for PKA. This work was supported by grants Wa463/9–5 and GRK837 from the Deutsche Forschungsgemeinschaft.     

A point mutation abolishes binding of cAMP to site A in the regulatory subunit of cAMP-dependent protein kinase

Each regulatory subunit of cAMP-dependent protein kinase has two tandem cAMP-binding sites, A and B, at the carboxyl terminus. Based on sequence homologies with the cAMP-binding domain of the Escherichia coli catabolite gene activator protein, a model has been constructed for each cAMP-binding domain. Two of the conserved features of each cAMP-binding site are an arginine and a glutamic acid which interact with the negatively charged phosphate and with the 2'-OH on the ribose ring, respectively. In the type I regulatory subunit, this arginine in cAMP binding site A is Arg-209. Recombinant DNA techniques have been used to change this arginine to a lysine. The resulting protein binds cAMP with a high affinity and associates with the catalytic subunit to form holoenzyme. The mutant holoenzyme also is activated by cAMP. However, the mutant R-subunit binds only 1 mol of cAMP/R-monomer. Photoaffinity labeling confirmed that the mutant R-subunit has only one functional cAMP-binding site. In contrast to the native R-subunit which is labeled at Trp-260 and Tyr-371 by 8-N3cAMP, the mutant R-subunit is convalently modified at a single site, Tyr-371, which correlates with a functional cAMP-binding site B. The lack of functional cAMP-binding site A also was confirmed by activating the mutant holoenzyme with analogs of cAMP which have a high specificity for either site A or site B. 8-NH2-methyl cAMP which preferentially binds to site B was similar to cAMP in its ability to activate both mutant and wild type holoenzyme whereas N6-monobutyryl cAMP, a site A-specific analog, was a very poor activator of the mutant holoenzyme. The results support the conclusions that 1) Arg-209 is essential for cAMP binding to site A and 2) cAMP binding to domain A is not essential for dissociation of the mutant holoenzyme.     

Phosphorylation of the p220 subunit of eIF-4F by cAMP dependent protein kinase and protein kinase C in vitro

Changes in the extent of phosphorylation of the 25 kDa subunit of eIF-4F occur during several major biological events including mitosis and heat shock in mammalian cells and shortly after fertilization of sea urchin (Lytechinus pictus) eggs. In vitro phosphorylation studies using highly purified protein kinases demonstrated that the 220 kDa subunit of eIF-4F was phosphorylated by cAMP dependent protein kinase, protein kinase C and probably to a lesser extent by cGMP dependent protein kinase. In addition, eIF-4A was readily phosphorylated by cAMP and cGMP dependent protein kinases whereas p48 of eIF-4F was not. The effect of these phosphorylation events on eIF-4F function, its assembly or disassembly, susceptibility to viral initiated proteolysis or the ability of p25 to be phosphorylated at serine-53 remain to be investigated.     

ATR-dependent phosphorylation of DNA-dependent protein kinase catalytic subunit in response to UV-induced replication stress

Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) upon ionizing radiation (IR) is essential for cellular radioresistance and nonhomologous-end-joining-mediated DNA double-strand break repair. In addition to IR induction, we have previously shown that DNA-PKcs phosphorylation is increased upon camptothecin treatment, which induces replication stress and replication-associated double-strand breaks. To clarify the involvement of DNA-PKcs in this process, we analyzed DNA-PKcs phosphorylation in response to UV irradiation, which causes replication stress and activates ATR (ATM-Rad3-related)/ATM (ataxia-telangiectasia mutated) kinases in a replication-dependent manner. Upon UV irradiation, we observed a rapid DNA-PKcs phosphorylation at T2609 and T2647, but not at S2056, distinct from that induced by IR. UV-induced DNA-PKcs phosphorylation occurs specifically only in replicating cells and is dependent on ATR kinase. Inhibition of ATR activity via caffeine, a dominant-negative kinase-dead mutant, or RNA interference led to the attenuation of UV-induced DNA-PKcs phosphorylation. Furthermore, DNA-PKcs associates with ATR in vivo and is phosphorylated by ATR in vitro, suggesting that DNA-PKcs could be the direct downstream target of ATR. Taken together, these results strongly suggest that DNA-PKcs is required for the cellular response to replication stress and might play an important role in the repair of stalled replication forks.     

Merlin links to the cAMP neuronal signaling pathway by anchoring the RIbeta subunit of protein kinase A

The cAMP-protein kinase A (PKA) pathway, important in neuronal signaling, is regulated by molecules that bind and target PKA regulatory subunits. Of four regulatory subunits, RIbeta is most abundantly expressed in brain. The RIbeta knockout mouse has defects in hippocampal synaptic plasticity, suggesting a role for RIbeta in learning and memory-related functions. Molecules that interact with or regulate RIbeta are still unknown. We identified the neurofibromatosis 2 tumor suppressor protein merlin (schwannomin), a molecule related to the ezrin-radixin-moesin family of membrane-cytoskeleton linker proteins, as a binding partner for RIbeta. Merlin and RIbeta demonstrated a similar expression pattern in central nervous system neurons and an overlapping subcellular localization in cultured hippocampal neurons and transfected cells. The proteins were coprecipitated from brain lysates by cAMP-agarose and coimmunoprecipited from cellular lysates with specific antibodies. In vitro binding studies verified that the interaction is direct. The interaction appeared to be under conformational regulation and was mediated via the alpha-helical region of merlin. Sequence comparison between merlin and known PKA anchoring proteins identified a conserved alpha-helical PKA anchoring protein motif in merlin. These results identify merlin as the first neuronal binding partner for PKA-RIbeta and suggest a novel function for merlin in connecting neuronal cytoskeleton to PKA signaling.     

Deletion of type IIalpha regulatory subunit delocalizes protein kinase A in mouse sperm without affecting motility or fertilization.

Cyclic AMP stimulates sperm motility in a variety of mammalian species, but the molecular details of the intracellular signaling pathway responsible for this effect are unclear. The type IIalpha isoform of protein kinase A (PKA) is induced late in spermatogenesis and is thought to localize PKA to the flagellar apparatus where it binds cAMP and stimulates motility. A targeted disruption of the type IIalpha regulatory subunit (RIIalpha) gene allowed us to examine the role of PKA localization in sperm motility and fertility. In wild type sperm, PKA is found primarily in the detergent-resistant particulate fraction and localizes to the mitochondrial-containing midpiece and the principal piece. In mutant sperm, there is a compensatory increase in RIalpha protein and a dramatic relocalization of PKA such that the majority of the holoenzyme now appears in the soluble fraction and colocalizes with the cytoplasmic droplet. Unexpectedly the RIIalpha mutant mice are fertile and have no significant changes in sperm motility. Our results demonstrate that the highly localized pattern of PKA seen in mature sperm is not essential for motility or fertilization.     

Regulation of AMP-activated protein kinase by multisite phosphorylation in response to agents that elevate cellular cAMP

The AMP-activated protein kinase (AMPK) and cAMP signaling systems are both key regulators of cellular metabolism. In this study, we show that AMPK activity is attenuated in response to cAMP-elevating agents through modulation of at least two of its alpha subunit phosphorylation sites, viz. alpha-Thr(172) and alpha1-Ser(485)/alpha2-Ser(491), in the clonal beta-cell line INS-1 as well as in mouse embryonic fibroblasts and COS cells. Forskolin, isobutylmethylxanthine, and the glucose-dependent insulinotropic peptide inhibited AMPK activity and reduced phosphorylation of the activation loop alpha-Thr(172) via inhibition of calcium/calmodulin-dependent protein kinase kinase-alpha and -beta, but not LKB1. These agents also enhanced phosphorylation of alpha-Ser(485/491) by the cAMP-dependent protein kinase. AMPK alpha-Ser(485/491) phosphorylation was necessary but not sufficient for inhibition of AMPK activity in response to forskolin/isobutylmethylxanthine. We show that AMPK alpha-Ser(485/491) can be a site for autophosphorylation, which may play a role in limiting AMPK activation in response to energy depletion or other regulators. Thus, our findings not only demonstrate cross-talk between the cAMP/cAMP-dependent protein kinase and AMPK signaling modules, but also describe a novel mechanism by which multisite phosphorylation of AMPK contributes to regulation of its enzyme activity.     

Spatial targeting of type II protein kinase A to filopodia mediates the regulation of growth cone guidance by cAMP

The second messenger cyclic adenosine monophosphate (cAMP) plays a pivotal role in axonal growth and guidance, but its downstream mechanisms remain elusive. In this study, we report that type II protein kinase A (PKA) is highly enriched in growth cone filopodia, and this spatial localization enables the coupling of cAMP signaling to its specific effectors to regulate guidance responses. Disrupting the localization of PKA to filopodia impairs cAMP-mediated growth cone attraction and prevents the switching of repulsive responses to attraction by elevated cAMP. Our data further show that PKA targets protein phosphatase-1 (PP1) through the phosphorylation of a regulatory protein inhibitor-1 (I-1) to promote growth cone attraction. Finally, we find that I-1 and PP1 mediate growth cone repulsion induced by myelin-associated glycoprotein. These findings demonstrate that the spatial localization of type II PKA to growth cone filopodia plays an important role in the regulation of growth cone motility and guidance by cAMP.     

Correlation of photolabeling with occupancy of cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I

Each regulatory subunit of the cAMP-dependent protein kinase contains two in-tandem cAMP binding sites. Photolabeling of holoenzyme I with 8-azidoadenosine 3',5'-monophosphate (8-N3-cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371. In order to correlate photolabeling of these two residues with occupancy of each specific cAMP binding site, photolabeling was carried out in the presence of various analogues of cAMP that bind preferentially to one site. Photolabeling of holoenzyme I after dissociation of 60% of 8-N3-[3H]cAMP with an excess of N6-monobutyryl-cAMP nearly abolished the incorporation of 8-N3-cAMP into Trp-260, whereas the modification of Tyr-371 was reduced by 49%. When 8-N3-[32P]cAMP was bound under equilibrium conditions in the presence of various cAMP analogues, N6-monobutyryl-cAMP also selectively abolished incorporation of radioactivity into Trp-260, whereas 8-(methylamino)-cAMP preferentially reduced the covalent modification of Tyr-371. Photolabeling with trace amounts of 8-N3-[32P]cAMP in the presence of saturating amounts of N6-monobutyryl-cAMP led to the covalent modification of only Tyr-371. In addition, photolabeling of Tyr-371 was enhanced synergistically in the presence of N6-monobutyryl-cAMP. MgATP reduced the covalent modification of both Trp-260 and Tyr-371 but showed no selectivity for either site. These studies support a model that correlates photolabeling of Trp-260 with occupancy of cAMP binding site A and photolabeling of Tyr-371 with occupancy of cAMP binding site B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biphasic response to 3',5'-cyclic adenosine monophosphate (cAMP) at the messenger ribonucleic acid level for a regulatory subunit of cAMP-dependent protein kinase

In the present study we have examined the effect of long-term stimulation with (Bu)2cAMP on mRNA levels for the hormone responsive regulatory subunit (RII beta) of cAMP-dependent protein kinase in cultured rat Sertoli cells. The effects of the same treatment on two other mRNAs [androgen binding protein (ABP) and cellular retinol binding protein (cRBP)], shown to be regulated by cAMP, were examined simultaneously. The addition of (Bu)2cAMP (0.1 mM) to primary Sertoli cell cultures, for 14 and 24 h, caused a 50- to 60-fold stimulation in the steady state levels of mRNA for RII beta. During the same period of stimulation, we also observed a significant increase (2- to 3-fold) in the mRNA levels for ABP, and a 80% decrease in the mRNA levels for cRBP. Continued stimulation for 36 and 48 h was associated with a significant time-dependent decrease in the mRNA level for RII beta, in spite of the continuous presence of (Bu)2cAMP (0.1 mM) in the medium. This reduced response by long term stimulation with (Bu)2cAMP appears to be specific for RII beta, since mRNA for ABP remained elevated and mRNA for cRBP remained depressed during the entire period of cAMP stimulation. Our data demonstrate the presence of a biphasic type of regulation at the mRNA level, specific for the regulatory subunit RII beta of cAMP-dependent protein kinase. This response may be analogous to the desensitization mechanisms observed at other levels of the cAMP signalling pathway. For proteins constituting part of the signal transduction pathway this type of biphasic regulation, may be particularly important in maintaining homeostasis in the cell.     

Oncogenic ras mediates apoptosis in response to protein kinase C inhibition through the generation of reactive oxygen species

Ras is a well established modulator of apoptosis. Suppression of protein kinase C (PKC) activity can selectively induce apoptosis in cells expressing a constitutively activated Ras protein. We wished to determine whether reactive oxygen species serve as an effector of Ras-mediated apoptosis. Ras-transformed NIH/3T3 cells contained higher basal levels of intracellular H(2)O(2) compared with normal NIH/3T3 cells, and PKC inhibition up-regulated ROS to 5-fold greater levels in Ras-transformed cells than in normal cells. Treatment with N-acetyl-l-cysteine reduced both the basal and inducible levels of intracellular H(2)O(2) in NIH/3T3-Ras cells and antagonized the induction of apoptosis by PKC inhibition. Culturing NIH/3T3-Ras cells in low oxygen conditions, which prevents ROS generation, also inhibited the apoptotic response to PKC inhibition. These results suggest that reactive oxygen species are necessary as downstream effectors of the Ras-mediated apoptotic response to PKC inhibition. However, the generation of ROS alone is not sufficient to induce apoptosis in Ras-transformed cells because inhibition of cell cycle progression prevented the induction of apoptosis in NIH/3T3-Ras cells without inhibiting the generation of intracellular H(2)O(2) observed after PKC inhibition. These findings suggest that continued cell cycle progression of Ras-transformed cells during PKC inhibition is also necessary for the induction of apoptosis.     

Acute phase mediator oncostatin M regulates affinity of alpha1-protease inhibitor for concanavalin A in hepatoma-derived but not lung-derived epithelial cells

Quantitative changes in plasma protein concentrations during tissue injury or inflammation (acute phase response) are often accompanied by specific alterations in the carbohydrate moieties of these proteins. The glycosylation changes comprise alterations in the type of branching of the carbohydrate structures as revealed by modulated reactivity of acute phase glycoproteins with the lectin concanavalin A. Interestingly, inflammation-induced changes in the glycosylation of acute phase proteins have been shown to affect the functional properties of these proteins. In this study we demonstrate that synthesis of acute phase protein alpha(1)-PI, the controlling inhibitor of neutrophil elastase, is significantly up-regulated in hepatic and lung-derived epithelial cells by the inflammatory mediator oncostatin M. Although oncostatin M markedly altered the concanavalin A reactivity of hepatic alpha(1)-PI, lung-derived epithelial cells did not change the pattern of alpha(1)-PI glycan branching upon stimulation with oncostatin M. These results indicate that inflammation-induced changes in glycosylation of alpha(1)-PI may have different impacts on functional properties of liver and lung-synthesized alpha(1)-PI.     

Tyrosine-371 contributes to the positive cooperativity between the two cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I

The regulatory (R) subunit of cAMP-dependent protein kinase I has been expressed in Escherichia coli, and oligonucleotide-directed mutagenesis was initiated in order to better understand structural changes that are induced as a consequence of cAMP-binding. Photoaffinity labeling of the type I holoenzyme with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371 [Bubis, J., & Taylor, S.S. (1987) Biochemistry 26, 3478-3486]. The site that was targeted for mutagenesis was Tyr-371. The intention was to establish whether the interactions between the tyrosine ring and the adenine ring of cAMP are primarily hydrophobic in nature or whether the hydroxyl group is critical for cAMP binding and/or for inducing conformational changes. A single base change converted Tyr-371 to Phe. This yielded an R subunit that reassociated with the catalytic subunit to form holoenzyme and bound 2 mol of cAMP/mol of R monomer. The cAMP binding properties of the holoenzyme that was formed with this mutant R subunit, however, were altered: (a) the apparent Kd(cAMP) was shifted from 16 to 60 nM; (b) Scatchard plots showed no cooperativity between the cAMP binding sites in the mutant in contrast to the positive cooperativity that is observed for the wild-type holoenzyme; (c) the Hill coefficient of 1.6 for the wild-type holoenzyme was reduced to 0.99. The Ka's for activation by cAMP were altered in the mutant holoenzyme in a manner that was proportional to the shift in Kd(cAMP).(ABSTRACT TRUNCATED AT 250 WORDS)