Signal transduction through the cAMP-dependent protein kinase

Molecular and Cellular Biochemistry - Temporal cellular events responsible for hormonal activation of responses mediated by the cAMP-dependent protein kinase (PKA) have been studied in living...     

Signal transduction by MAP kinase cascades in budding yeast

Budding yeast contain at least four distinct MAPK (mitogen activated protein kinase) cascades that transduce a variety of intracellular signals: mating-pheromone response, pseudohyphal/invasive growth, cell wall integrity, and high osmolarity adaptation. Although each MAPK cascade contains a conserved set of three protein kinases, the upstream activation mechanisms for these cascades are diverse, including a trimeric G protein, monomeric small G proteins, and a prokaryotic-like two-component system. Recently, it became apparent that there is extensive sharing of signaling elements among the MAPK pathways; however, little undesirable cross-talk occurs between various cascades. The formation of multi-protein signaling complexes is probably centrally important for this insulation of individual MAPK cascades.     

Signal transduction of constitutively active protein kinase C epsilon

The protein kinase C (PKC) family is the most prominent target of tumor-promoting phorbol esters. For the PKCε isozyme, different intracellular localizations and oncogenic potential in several but not all experimental systems have been reported. To obtain information about PKCε-signaling, we investigated the effects of constitutively active rat PKCε (PKCεA/E, alanine 159 is replaced by glutamic acid) in HeLa cells in a doxycycline-inducible vector. Upon induction of PKCεA/E expression by doxycycline, the major part of PKCεA/E was localized to the Golgi. This led (i) to phosphorylations of PKCε^S729, Elk-1^S383, PDK1^S241 and Rb^S807/S811, (ii) to elevated expression of receptor of activated C kinase 2 (RACK2) after 12 h, and (iii) increased colony formation in soft agar, increased cell migration and invasion, but not to decreased doubling time. Following induction of PKCεA/E-expression by doxycycline for 24 h and additional short-term treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), PKCεA/E translocated to the plasma membrane and increased phosphorylation of MARCKS^S152/156. Treatment with doxycycline/TPA or TPA alone increased phosphorylations of Elk-1^S383, PDK1^S241, Rb^S807/S811, PKCδ^T505, p38MAPK^T180/Y182, MEK1/2^S217/S221 and ERK2^T185/T187. MARCKS was not phosphorylated after treatment with TPA alone, demonstrating that in this system it is phosphorylated only by PKCε localized to the plasma membrane but not by PKCα or δ, the other TPA-responsive PKC isozymes in HeLa cells. These results demonstrate that PKCε can induce distinctly different signaling from the Golgi and from the plasma membrane.     

Signal transduction through the T cell receptor is dynamically regulated by balancing kinase and phosphatase activities

Within seconds of T cell receptor engagement, a well-characterized series of tyrosine phosphorylation events mediate cellular activation. It is widely accepted that these initial phosphorylations remain stable until protein tyrosine phosphatases return the cell to its basal level. Based on a model of peripheral blood T cell activation, in which the kinetics of phosphorylation can be modulated, we propose an alternate hypothesis that T cell signaling is highly dynamic. Our results demonstrate that tyrosine phosphorylation and dephosphorylation are occurring co-temporally after T cell receptor cross-linking, regulated by a delicate balance of kinases and phosphatases.     

Signal transduction within the nucleus by mitogen-activated protein kinase.

The nucleus is an important target of signal transduction by growth factor receptors that stimulate mitogen-activated protein (MAP) kinases. We tested the hypothesis that MAP kinases have a signaling role within the nucleus by examining the effect of the expression of a human MAP kinase isoform (p41mapk) in tissue culture cells. The expressed p41mapk was found to be localized in both the cytoplasmic and nuclear compartments of the cells. Significantly, the expression of p41mapk caused an increase in the phosphorylation of a nuclear substrate: Ser62 of c-Myc. Phosphorylation at Ser62 stimulated the activity of the NH2-terminal transactivation domain of c-Myc. Thus, p41mapk causes the phosphorylation and regulation of a physiologically significant nuclear target of signal transduction. These data establish that at least one MAP kinase isoform has a nuclear role during signal transduction.     

Signal transduction through integrins: A central role for focal adhesion kinase?

The integrins are receptors for proteins of the extracellular matrix, both providing a physical link to the cytoskeleton and transducing signals from the extracellular matrix. Activation of integrins leads to tyrosine and serine phosphorylation of a number of proteins, elevation of cytosolic calcium levels, cytoplasmic alkalinization, changes in phospholipid metabolism and, ultimately, changes in gene expression. The recently discovered focal adhesion kinase localizes to focal contacts, which are sites of integrin clustering, and focal adhesion kinase can physically associate with integrins in vitro. As integrins lack intrinsic catalytic activity, focal adhesion kinase is a candidate for a signaling molecule that is recruited by integrins in order to trigger the generation of intracellular second messengers. Thus, focal adhesion kinase may play a central role in signal transduction through integrins.     

Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae

Chlamydia pneumoniae is an important respiratory pathogen. Recently, its presence has been demonstrated in atherosclerotic lesions. In this study, we characterized C. pneumoniae-mediated activation of endothelial cells and demonstrated an enhanced expression of endothelial adhesion molecules followed by subsequent rolling, adhesion, and transmigration of leukocytes (monocytes, granulocytes). These effects were blocked by mAbs against endothelial and/or leukocyte adhesion molecules (beta1 and beta2 integrins). Additionally, activation of different signal transduction pathways in C. pneumoniae-infected endothelial cells was shown: protein tyrosine phosphorylation, up-regulation of phosphorylated p42/p44 mitogen-activated protein kinase, and NF-kappaB activation/translocation occurred within 10-15 min. Increased mRNA and surface expression of E-selectin, ICAM-1, and VCAM-1 were noted within hours. Thus, C. pneumoniae triggers a cascade of events that could lead to endothelial activation, inflammation, and thrombosis, which in turn may result in or may promote atherosclerosis.     

Signal transduction pathways activated by chorionic gonadotropin in the primate endometrial epithelial cells

Successful implantation requires synergism between the developing embryo and the receptive endometrium. In the baboon, infusion of chorionic gonadotropin (CG) modulates both morphology and physiology of the epithelial and stromal cells of the receptive endometrium. This study explored the signal transduction pathways activated by CG in endometrial epithelial cells from baboon (BE) and human (HES). Incubations of BE and HES cells with CG did not significantly alter adenylyl cyclase activity or increase intracellular cAMP when compared with Chinese hamster ovarian cells stably transfected with the full-length human CG/luteinizing hormone (LH) receptor (CHO-LH cells). However, in BE and HES cells, CG induced the phosphorylation of several proteins, among them, extracellular signal-regulated protein kinases 1 and 2 (ERK 1/2). Phosphorylation of ERK 1/2 in uterine epithelial cells was protein kinase A (PKA) independent. This novel signaling pathway is functional because, in response to CG stimulation, prostaglandin E(2) (PGE(2)) was released into the media and increased significantly 2 h following CG stimulation. CG-stimulated PGE(2) synthesis in epithelial cells was inhibited by a specific mitogen-activated protein kinase (MEK 1/2) inhibitor, PD 98059. In conclusion, immediate signal transduction pathways induced by CG in endometrial epithelial cells are cAMP independent and stimulate phosphorylation of ERK 1/2 via a MEK 1/2 pathway, leading to an increase in PGE(2) release as the possible result of cyclooxygenase-2 activation.     

Signal transduction of angiotensin II type 1 receptor through receptor tyrosine kinase

In cultured vascular smooth muscle cells, the angiotensin II (AngII) type-1 (AT(1)) receptor generates growth-promoting signals via the epidermal growth factor (EGF) receptor system. This 'transactivation' mechanism now appears to be utilized by a variety of G-protein-coupled receptors in many cells. The AngII-induced EGF receptor transactivation leads to activation of downstream signaling molecules including Ras, ERK, c-fos, Akt/protein kinase B, and p70 S6 kinase. We propose three possible mechanisms may be involved in the transactivation, (i) an upstream tyrosine kinase, (ii) reactive oxygen species, and (iii) a juxtacrine activation of the EGF receptor ligand. Whether the EGF receptor signal transduction induced by AngII plays an essential role in cardiovascular remodeling remains to be investigated.     

Signal transduction in neurons: effects of cellular prion protein on fyn kinase and ERK1/2 kinase

Background

It has been reported that cellular prion protein (PrP^c) co-localizes with caveolin-1 and participates to signal transduction events by recruiting Fyn kinase. As PrP^c is a secreted protein anchored to the outer surface membrane through a glycosylphosphatidylinositol (GPI) anchor (^secPrP) and caveolin-1 is located in the inner leaflet of plasma membrane, there is a problem of how the two proteins can physically interact each other and transduce signals.

Results

By using the GST-fusion proteins system we observed that PrP^c strongly interacts with caveolin-1 scaffolding domain and with a caveolin-1 hydrophilic C-terminal region, but not with the caveolin-1 N-terminal region. In vitro binding experiments were also performed to define the site(s) of PrP^c interacting with cav-1. The results are consistent with a participation of PrP^c octapeptide repeats motif in the binding to caveolin-1 scaffolding domain. The caveolar localization of PrP^c was ascertained by co-immunoprecipitation, by co-localization after flotation in density gradients and by confocal microscopy analysis of PrP^c and caveolin-1 distributions in a neuronal cell line (GN11) expressing caveolin-1 at high levels.

Conclusions

We observed that, after antibody-mediated cross-linking or copper treatment, PrP^c was internalized probably into caveolae. We propose that following translocation from rafts to caveolae or caveolae-like domains, ^secPrP could interact with caveolin-1 and induce signal transduction events.

     

Signal transduction pathways regulating Rho-mediated stress fibre formation: requirement for a tyrosine kinase.

Lysophosphatidic acid (LPA) and bombesin rapidly stimulate the formation of focal adhesions and actin stress fibres in serum-starved Swiss 3T3 fibroblasts, a process regulated by the small GTP binding protein Rho. To investigate further the signalling pathways leading to these responses, we have tested the roles of three intracellular signals known to be induced by LPA: activation of protein kinase C (PK-C), Ca2+ mobilization and decreased cAMP levels. Neither PK-C activation nor increased [Ca2+]i, alone or in combination, induced stress fibre formation, and in fact activators of PK-C inhibited this response to LPA and bombesin. The G(i)-mediated decrease in cAMP was not required for the response to LPA, and increased cAMP levels did not prevent stress fibre formation. In contrast, the tyrosine kinase inhibitor genistein inhibited the formation of stress fibres induced by both extracellular factors and microinjected Rho protein. Genistein also inhibited the Rho-dependent clustering of phosphotyrosine-containing proteins at focal adhesions, and the increased tyrosine phosphorylation of several proteins including pp125FAK, induced by LPA and bombesin. This suggests a model where Rho-induced activation of a tyrosine kinase is required for the formation of stress fibres.