A novel regulatory mechanism of MAP kinases activation and nuclear translocation mediated by PKA and the PTP-SL tyrosine phosphatase

Protein tyrosine phosphatase PTP-SL retains mitogen-activated protein (MAP) kinases in the cytoplasm in an inactive form by association through a kinase interaction motif (KIM) and tyrosine dephosphorylation. The related tyrosine phosphatases PTP-SL and STEP were phosphorylated by the cAMP-dependent protein kinase A (PKA). The PKA phosphorylation site on PTP-SL was identified as the Ser(231) residue, located within the KIM. Upon phosphorylation of Ser(231), PTP-SL binding and tyrosine dephosphorylation of the MAP kinases extracellular signal-regulated kinase (ERK)1/2 and p38alpha were impaired. Furthermore, treatment of COS-7 cells with PKA activators, or overexpression of the Calpha catalytic subunit of PKA, inhibited the cytoplasmic retention of ERK2 and p38alpha by wild-type PTP-SL, but not by a PTP-SL S231A mutant. These findings support the existence of a novel mechanism by which PKA may regulate the activation and translocation to the nucleus of MAP kinases.     

Uncoupling of betaIIPKC from its targeting protein RACK1 in response to ethanol in cultured cells and mouse brain.

Protein kinase C (PKC) is involved in many neuroadaptive responses to ethanol in the nervous system. PKC activation results in translocation of the enzyme from one intracellular site to another. Compartmentalization of PKC isozymes is regulated by targeting proteins such as receptors for activated C kinase (RACKs). It is possible, therefore, that ethanol-induced changes in the function and compartmentalization of PKC isozymes could be due to changes in PKC targeting proteins. Here we study the response of the targeting protein RACK1 and its corresponding kinase betaIIPKC to ethanol, and propose a novel mechanism to explain how ethanol modulates signaling cascades. In cultured cells, ethanol induces movement of RACK1 to the nucleus without affecting the compartmentalization of betaIIPKC. Ethanol also inhibits betaIIPKC translocation in response to activation. These results suggest that ethanol inhibition of betaIIPKC translocation is due to miscompartmentalization of the targeting protein RACK1. Similar events occurred in mouse brain. In vivo exposure to ethanol caused RACK1 to localize to nuclei in specific brain regions, but did not affect the compartmentalization of betaIIPKC. Thus, some of the cellular and neuroadaptive responses to ethanol may be related to ethanol-induced movement of RACK1 to the nucleus, thereby preventing the translocation and corresponding function of betaIIPKC.     

Human Tau may Modify Glucocorticoids-Mediated Regulation of cAMP-dependent Kinase and Phosphorylated cAMP Response Element Binding Protein

Phosphorylation of the cAMP response element binding protein (CREB) by cAMP-dependent kinase (PKA) is critical to memory formation. However, activation of PKA can also increase tau phosphorylation, which may contribute to memory impairment. Therefore, the regulation of PKA may be part of the mechanism by which glucocorticoids (GCs) influence memory. Additionally, the cellular response to GCs may be affected by the presence of human tau. The goal of this paper was to study GCs-mediated regulation of PKA as well as CREB and tau phosphorylation in wild-type HEK293 cells and HEK293 cells stably expressing human tau441 (HEK293/tau441 cells). By using dexamethasone (DEX) as GCs, we found that DEX induced a tau-dependent selective decrease in the level of PKA RIIβ subunit protein. The observed decrease in RIIβ expression was not due to alterations of mRNA levels and was reversed by inhibiting the proteasome with lactacystin. Moreover, the decrease in RIIβ did not diminish the co-localization of the catalytic subunit of PKA with tau and might contribute to the DEX-induced increase in tau phosphorylation at Ser-214. DEX also induced a tau-dependent decrease in CREB phosphorylation that could not be reversed by activating PKA with forskolin. Taken together, these results show that human tau protein may alter the GCs-mediated regulation of PKA activity and CREB phosphorylation.     

Nuclear activation and translocation of mitogen-activated protein kinases modulated by ethanol in embryonic liver cells

Activation and nuclear translocation of mitogen activated protein (MAP) kinases in ethanol-treated embryonic liver cells (BNLCL2) was investigated. The relative amount of MAPK proteins, MAP kinase activity and MAPK/LDH (lactate dehydrogenase) ratios were determined in nuclear and cytosolic fractions before and after serum stimulation. In ethanol-treated cells, serum-stimulated MAPK activation was potentiated in both cytosolic and nuclear fractions. Levels of both the p42 and p44 MAPK proteins increased in nuclear fractions from cells treated with ethanol alone for 24 h. Serum-stimulated nuclear translocation of both p42 and p44 MAPK was potentiated in ethanol-treated cells. Nuclear fractions from ethanol-treated cells had a modest increase in MAP kinase activity concurrent with the increased MAPK protein levels. The ratio of MAPK/LDH increased in nuclear fractions with increasing concentrations of ethanol and after serum stimulation. This further confirmed the nuclear translocation of MAPK and also demonstrated that it is not a non-specific effect of ethanol. These results demonstrate, for the first time, that in BNLCL2 liver cells ethanol treatment has dual effects. First, ethanol triggered nuclear translocation of MAPK without causing its activation. Second, it potentiated serum-stimulated activation and translocation of MAPK in the nucleus. These findings provide a novel mechanism through which ethanol may affect cellular and nuclear processes in liver cells.     

Acute ethanol intake induces mitogen-activated protein kinase activation,platelet-derived growth factor receptor phosphorylation,and oxidative stress in resistance arteries

In the present study, we investigated the role of angiotensin type I (AT₁) receptor in reactive oxygen species (ROS) generation and mitogen-activated protein kinases (MAPK) activation induced by acute ethanol intake in resistance arteries. We also evaluated the effect of ethanol on platelet-derived growth factor receptors (PDGF-R) phosphorylation and the role of this receptor on ROS generation by ethanol. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. Acute ethanol intake did not alter angiotensin I or angiotensin II levels in the rat mesenteric arterial bed (MAB). Ethanol induced vascular oxidative stress, and this response was not prevented by losartan (10 mg/kg; p.o. gavage), a selective AT₁ receptor antagonist. MAB from ethanol-treated rats displayed increased SAPK/JNK and PDGF-R phosphorylation, responses that were not prevented by losartan. The phosphorylation levels of protein kinase B (Akt) and eNOS were not affected by acute ethanol intake. MAB nitrate levels and the reactivity of this tissue to acetylcholine, phenylephrine, and sodium nitroprusside were not affected by ethanol intake. Ethanol did not alter plasma antioxidant capacity, the levels of reduced glutathione, or the activities of superoxide dismutase and catalase in the rat MAB. Short-term effects of ethanol (50 mmol/l) were evaluated in vascular smooth muscle cells (VSMC) isolated from rat MAB. Ethanol increased ROS generation, and this response was not affected by AG1296, a PDGF-R inhibitor, or losartan. Finally, ethanol did not alter MAPK or PDGF-R phosphorylation in cultured VSMC. Our study provides novel evidence that acute ethanol intake induces ROS generation, PDGF-R phosphorylation, and MAPK activation through AT(1)-independent mechanisms in resistance arteries in vivo. MAPK and PDGF-R play a role in vascular signaling and cardiovascular diseases and may contribute to the vascular pathobiology of ethanol.     

Ethanol induces apoptosis in hepatocytes by a pathway involving novel protein kinase C isoforms

Ethanol abuse is one of the major etiologies of cirrhosis. Ethanol has been shown to induce apoptosis via activation of oxidative stress, mitogen-activated protein kinases (MAPK), and tyrosine kinases. However, there is a paucity of data that examine the interplay among these molecules. In the present study we have systematically elucidated the role of novel protein kinase C isoforms (nPKC; PKCdelta and PKCepsilon) in ethanol-induced apoptosis in hepatocytes. Ethanol enhanced membrane translocation of PKCdelta and PKCepsilon, which was associated with the phosphorylation of p38MAPK, p42/44MAPK and JNK1/2, and the nuclear translocation of NF-kappaB and AP-1. This resulted in increased apoptosis in primary rat hepatocytes. Inhibition of both PKCdelta and PKCepsilon resulted in a decreased MAPK activation, decreased nuclear translocation of NF-kappaB and AP-1, and inhibition of apoptosis. In addition, ethanol activated the tyrosine phosphorylation of PKCdelta via tyrosine kinase in hepatocytes. The tyrosine phosphorylated PKCdelta was cleaved by caspase-3 and these fragments were translocated to the nucleus. Inhibition of ethanol-induced oxidative stress blocked the membrane translocation of PKCdelta and PKCepsilon, and the tyrosine phosphorylation of PKCdelta in hepatocytes. Inhibition of oxidative stress, tyrosine kinase or caspase-3 activity caused a decreased nuclear translocation of PKCdelta in response to ethanol, and was associated with less apoptosis. Conclusion: These results provide a newly-described mechanism by which ethanol induces apoptosis via activation of nPKC isoforms in hepatocytes.     

Modulation of F-actin rearrangement by the cyclic AMP/cAMP-dependent protein kinase (PKA) pathway is mediated by MAPK-activated protein kinase 5 and requires PKA-induced nuclear export of MK5

The MAPK-activated protein kinases belong to the Ca2+/calmodulin-dependent protein kinases. Within this group, MK2, MK3, and MK5 constitute three structurally related enzymes with distinct functions. Few genuine substrates for MK5 have been identified, and the only known biological role is in ras-induced senescence and in tumor suppression. Here we demonstrate that activation of cAMP-dependent protein kinase (PKA) or ectopic expression of the catalytic subunit Calpha in PC12 cells results in transient nuclear export of MK5, which requires the kinase activity of both Calpha and MK5 and the ability of Calpha to enter the nucleus. Calpha and MK5, but not MK2, interact in vivo, and Calpha increases the kinase activity of MK5. Moreover, Calpha augments MK5 phosphorylation, but not MK2, whereas MK5 does not seem to phosphorylate Calpha. Activation of PKA can induce actin filament accumulation at the plasma membrane and formation of actin-based filopodia. We demonstrate that small interfering RNA-triggered depletion of MK5 interferes with PKA-induced F-actin rearrangement. Moreover, cytoplasmic expression of an activated MK5 variant is sufficient to mimic PKA-provoked F-actin remodeling. Our results describe a novel interaction between the PKA pathway and MAPK signaling cascades and suggest that MK5, but not MK2, is implicated in PKA-induced microfilament rearrangement.     

Apolipoprotein A-I induces translocation of protein kinase C[alpha] to a cytosolic lipid-protein particle in astrocytes

Apolipoprotein A-I (apoA-I) induces the translocation of newly synthesized cholesterol as well as caveolin-1 to the cytosolic lipid-protein particle (CLPP) fraction in astrocytes before its appearance in high density lipoprotein generated in the medium (Ito, J., Y. Nagayasu, K. Kato, R. Sato, and S. Yokoyama. 2002. Apolipoprotein A-I induces translocation of cholesterol, phospholipid, and caveolin-1 to cytosol in rat astrocytes. J. Biol. Chem. 277: 7929-7935). We here report the association of signal-related molecules with CLPP. ApoA-I induces rapid translocation of protein kinase Calpha to the CLPP fraction and its phosphorylation in astrocytes. ApoA-I also induces the translocation of phospholipase Cgamma to CLPP. Diacylglyceride (DG) production is increased by apoA-I in the cells, with a maximum at 5 min after the stimulation, and the increase takes place also in the CLPP fraction. An inhibitor of receptor-coupled phospholipase C, U73122, inhibited all the apoA-I-induced events, such as DG production, cholesterol translocation to the cytosol, release of cholesterol, and translocation of protein kinase Calpha into the CLPP fraction. CLPP may thus be involved in the apoA-I-initiated signal transduction in astrocytes that is related to intracellular cholesterol trafficking for the generation of high density lipoprotein in the brain.     

Regulation of lung epithelial cell morphology by cAMP-dependent protein kinase type I isozyme

Cell shape is mediated in part by the actin cytoskeleton and the actin-binding protein vinculin. These proteins in turn are regulated by protein phosphorylation. We assessed the contribution of cAMP-dependent protein kinase A isozyme I (PKA I) to lung epithelial morphology using the E10/E9 sibling cell lines. PKA I concentration is high in flattened, nontumorigenic E10 cells but low in their round E9 transformants. PKA I activity was lowered in E10 cells by stable transfection with a dominant negative RIalpha mutant of the PKA I regulatory subunit and was raised in E9 cells by stable transfection with a wild-type Calpha catalytic subunit construct. Reciprocal changes in morphology ensued. E10 cells became rounder and grew in colonies, their actin microfilaments were disrupted, and vinculin localization at cell-cell junctions was diminished. The converse occurred in E9 cells on elevating their PKA I content. Demonstration that PKA I is responsible for the dichotomy in these cellular behaviors suggests that manipulating PKA I concentrations in lung cancer would provide useful adjuvant therapy.     

Protein kinase A-mediated CREB phosphorylation is an oxidant-induced survival pathway in alveolar type II cells

Oxidant stress plays a role in the pathogenesis of pulmonary diseases, including fibrotic lung disease and cancer. We previously found that hydrogen peroxide (H₂O₂) initiates an increase in Ca²⁺/cAMP-response element binding protein (CREB) phosphorylation in C10 alveolar type II cells that requires activation of extracellular regulated kinases 1/2 (ERK1/2). Here, we investigated the role of crosstalk between protein kinase A (PKA) and epidermal growth factor receptor (EGFR) in oxidant-induced signaling to ERK1/2 and CREB in C10 cells. Application of H₂O₂ increased nuclear accumulation of PKA, and inhibition of PKA with H89 reduced oxidant-mediated phosphorylation of both CREB and ERK1/2. Single cell measurements of cAMP and redox status, using a FRET-based biosensor and a redox-sensitive GFP, respectively, indicated that H₂O₂ increases production of cAMP that correlates with redox state. Inhibition of EGFR activity decreased both H₂O₂-induced CREB phosphorylation and translocation of PKA to the nucleus, suggesting that crosstalk between PKA and EGFR underlies the oxidant-induced CREB response. Furthermore, knockdown of CREB expression using siRNA led to a decrease in bcl-2 and an increase in oxidant-induced apoptosis. Together these data reveal a novel role for crosstalk between PKA, ERK1/2 and CREB that mediates cell survival during oxidant stress.