Temporal analysis of PI-3kinase and MAPK signal transduction during skeletal muscle overload

Skeletal muscles can adapt to increased mechanical forces (or loading) by increasing the size and strength of the muscle. Knowledge of the molecular mechanisms by which muscle responds to increased loading may lead to the discovery of novel treatment strategies for muscle wasting and frailty. The objective of this research was to examine the temporal associations between the activation of specific signaling pathway intermediates and their potential upstream regulator(s) in response to increased muscle loading. Previous work has demonstrated that focal adhesion kinase (FAK) activity is increased in overloaded hypertrophying skeletal muscle. Thus FAK is a candidate for transducing the loading stimulus in skeletal muscle, potentially by activating phosphatidylinositol 3-kinase (PI3K) and members of the mitogen-activated protein kinase (MAPK) family. However, it was unknown if muscle overload would result in activation of PI3K or the MAPKs. Thus, this work seeks to characterized the temporal response of (1) MAPK phosphorylation (including Erk 2, p38 MAPK and JNK), (2) PI3K activity, and (3) FAK tyrosine phosphorylation in response to 24 hours of compensatory overload in the rat soleus and plantaris muscles. In both muscles, overload resulted in transient Increases in the phosphorylation state of Erk2 and JNK, which peaked within the first hour of overload and returned to baseline thereafter. In contrast, p38 MAPK phosphorylation remained elevated throughout the entire 24-hour overload period. Moreover, overload increased PI3K activity only, in the plantaris and only at 12 hours. Moreover, 24 hours of overload induced a significant increase in total protein content in the plantaris but not the soleus. Thus an increase in total muscle protein content within the 24-hour loading period was observed only in muscle exhibiting increased PI3K activity. Surprisingly, FAK tyrosine phosphorylation was not increased during the overload period in either muscle, indicating that PI3K activation and increased MAPK phosphorylation were independent of increased FAK tyrosine phosphorylation. In summary, increased PI3K activity and sustained elevation of p38 MAPK phosphorylation were associated with muscle overload, identifying these pathways as potential mediators of the early hypertrophic response to skeletal muscle overload. This suggests that stimuli or mechanisms that activate these pathways may reduce/minimize muscle wasting and frailty. ^

CARMA3 serves as a critical mediator of NF-kappaB in multiple signal transduction pathways

The central dogma of molecular biology dictates that DNA is transcribed into RNA, which is later translated into protein. One of the early activators in this process is the transcription factor NF-κB. We have determined that an NF-κB inducer, CARMA3, is required for proper neural tube closure, similar to other NF-κB inducers. Using a genetic knockout of CARMA3, we demonstrated that it is required for Gαq-coupled GPCR-induced NF-κB activation. This is facilitated through a MAPK and IKK phosphorylation-independent mechanism, most likely by controlling NEMO-associated ubiquitination. We have also shown that CARMA3 is required for EGF and HRG-induced NF-κB activation. This activation requires the activity of both EGFR and HER2, as well as PKC. Again, we observed no defect in IKK phosphorylation, although we determined a clear defect in IKK activation. Finally, we have begun to determine the role of CARMA3 to both EGFR and HER2-induced tumorigenicity. By overexpressing a constitutive active mutant of HER2 in our CARMA3 WT and KO MEF cells, we have shown CARMA3 is important for HER2-driven soft agar colony growth. We have also shown that knockdown of endogenous CARMA3 in the EGFR-overexpressing A431 cell line abolishes EGF-induced NF-κB activation. These same cells have a dramatically reduced capacity to form colonies in soft agar as well. Using both mouse xenografts and a transgenic model of HER2-induced breast cancer, we have initiated studies which will help to determine the role of CARMA3 to in vivo tumorigenesis. Collectively, this work reveals novel roles for the CARMA3 protein in development, GPCR and EGFR/HER2 signaling. It also suggests that CARMA3 is involved in EGFR/HER2 mediated tumorigenesis, possibly indicating a novel therapeutic target for use in treatment of cancer. ^

Signal transduction of {\it HER-2\/}/{\it neu\/} receptor tyrosine kinase

Overexpression and/or amplification of HER2/neu is frequently detected in many human cancers. Activation of p185 tyrosine kinase can be achieved by point mutation, overexpression, deletion, and heterodimerization with other class I receptors. In this study I investigated the signal transduction pathways mediating the oncogenic signal of the point mutation-activated rat p185. I demonstrated that tyrosine phosphorylation of Shc and formation of Shc/Grb2 complex correlated to the transformation of NIH3T3 cells caused by the point mutation-activated rat HER2/neu. Furthermore, I observed that association with Shc was severely impaired by deletion of most of the major autophosphorylation sites of the point-mutated p185. The truncated p185 product, however, fully retained its ability to transform NIH3T3 cells, induce Shc tyrosine phosphorylation and Shc/Grb2 complex formation. These results suggest that tyrosine phosphorylation of Shc which allows formation of Shc/Grb2 complex may play an important role in cell transformation induced by the point mutation-activated p185, and that stable binding to mutant p185 may not be necessary for Shc to mediate this signaling pathway.^ Recent studies have suggested that formation of the complex containing Sos, Grb2 and Shc is important in coupling receptor tyrosine kinases to the Ras signaling pathway. To clarify the role of this trimer in the oncogenic signaling of the activated p185, I set out to interfere with the protein-protein interactions in Shc/Grb2/Sos complex by introducing Grb2 mutants with deletions in either amino- ($\Delta$N-Grb2) or carboxyl- ($\Delta$C-Grb2) terminal SH3 domains into B104-1-1 cells derived from NIH3T3 cells that express the point mutation-activated HER-2/neu. I found that the transformed phenotypes of the B104-1-1 cells were largely reversed by expression of the $\Delta$N-Grb2. The effect of the $\Delta$C-Grb2 on phenotypic reversion was much weaker. Biochemical analysis showed that the $\Delta$N-Grb2 was able to associate Shc but not the activated p185 nor Sos, while the $\Delta$C-Grb2 bound to Shc, the activated p185, and Sos. The p185-mediated Ras activation was severely inhibited by the $\Delta$N-Grb2 but not the $\Delta$C-Grb2. Taken together, these data demonstrate that interruption of the interaction between Shc and the endogenous Grb2 by the $\Delta$N-Grb2 is able to impair the oncogenic signaling of the mutation-activated p185, indicating that (i) the $\Delta$N-Grb2 functions as a strong dominant-negative mutant, (ii) Shc/Grb2/Sos pathway plays a major role in mediating the oncogenic signal of the mutation-activated p185. Unlike the $\Delta$N-Grb2, the $\Delta$C-Grb2 appears to be a relatively weak dominant-negative mutant, probably due to its ability to largely fulfill the biological functions of the wild-type Grb2. ^

Heterotrimeric G protein -mediated signal transduction in Neurospora crassa

Heterotrimeric G protein-mediated signal transduction is one of numerous means that cells utilize to respond to external stimuli. G proteins consist of α, β andγ subunits. Extracellular ligands bind to seven-transmembrane helix receptors, triggering conformational changes. This is followed by activation of coupled G proteins through the exchange of GDP for GTP on the Gα subunit. Once activated, Gα-GTP dissociates from the βγ dimer. Both of these two moieties can interact with downstream effectors, such as adenylyl cyclase, phospholipase C, phosphodiesterases, or ion channels, leading to a series of changes in cellular metabolism and physiology. ^ Neurospora crassa is a eukaryotic multicellular filamentous fungus, with asexual/vegetative and sexual phases to its life cycle. Three Gα (GNA-1, GNA-2, GNA-3) and one Gβ (GNB-1) proteins have been identified in this organism. This dissertation investigates GNA-1 and GNB-1 mediated signaling pathways in N. crassa. ^ GNA-1 was the first identified microbial Gα that belongs to a mammalian superfamily (Gαi). Deletion of GNA-1 leads to multiple defects in N. crassa. During the asexual cycle, Δgna-1 strains display a slower growth rate and delayed conidiation on solid medium. In the sexual cycle, the Δgna-1 mutant is male-fertile but female-sterile. Biochemical studies have shown that Δ gna-1 strains have lower adenosine 3′–5 ′ cyclic monophosphate (cAMP) levels than wild type under conditions where phenotypic defects are observed. In this thesis work, strains containing one of two GTPase-deficient gna-1 alleles (gna-1 R178C, gna-1Q204L) leading to constitutive activation of GNA-1 have been constructed and characterized. Activation of GNA-1 causes uncontrolled aerial hyphae proliferation, elevated sensitivity to heat and oxidative stresses, and lower carotenoid synthesis. To further study the function of GNA-1, constructs to enable expression of mammalian Gαi superfamily members were transformed into a Δ gna-1 strain, and complementation of Δgna-1 defects investigated. Gαs, which is not a member of Gα i superfamily was used as a control. These mammalian Gα genes were able to rescue the vegetative growth rate defect of the Δ gna-1 strain in the following order: Gαz > Gα o > Gαs > Gαt > Gαi. In contrast, only Gαo was able to complement the sexual defect of a Δgna-1 strain. With regard to the thermotolerance phenotype, none of the mammalian Gα genes restored the sensitivity to a wild type level. These results suggest that GNA-1 regulates two independent pathways during the vegetative and sexual cycles in N. crassa. ^ GNB-1, a G protein β subunit from N. crassa, was identified and its functions investigated in this thesis work. The sequence of the gnb-1 gene predicts a polypeptide of 358 residues with a molecular mass of 39.7 kDa. GNB-1 exhibits 91% identity to Cryphonectria parasitica CPGB-1, and also displays significant homology with human and Dictyostelium Gβ genes (∼66%). A Δ gnb-1 strain was constructed and shown to exhibit defects in asexual spore germination, vacuole number and size, mass accumulation and female fertility. A novel role for GNB-1 in regulation of GNA-1 and GNA-2 protein levels was also demonstrated. ^

The SasS -SasR two-component signal transduction system required for early Myxococcus xanthus multicellular *development

Initiation of Myxococcus xanthus multicellular development requires both nutrient limitation and high cell density. The extracellular signal, A signal, which consists of a set of amino acids at specific concentrations, serves as a cell density signal in M. xanthus early development. A reporter gene, designated 4521, that requires both starvation and A signal for developmental expression was used to identify mutations in the signal transduction pathways. A group of point mutations located in the chromosomal sasB locus that bypasses both requirements was previously isolated. One of these point mutations, sasB7, was mapped to the sasS gene, which is predicted to encode a transmembrane histidine protein kinase required for normal development. SasS is a positive regulator of 4521 and a candidate A signal sensor. This dissertation continues the characterization of the sasB locus, focusing on the sasR gene and the functional relationship of SasS and SasR. ^ The sasR gene is located 2.2-kb downstream of sasS. It is predicted to encode an NtrC-like response regulator, which belongs to the family of sigma54 transcriptional activators. SasR is a positive regulator of 4521 gene and is required for normal development. The sasR mutant displays phenotypes similar to that of sasS mutant. Both SasS and SasR are required for the A-signal-dependent 4521 expression. Genetic epistasis analysis indicates that SasR functions downstream of SasS. Biochemical studies show that SasS has autokinase activity, and phosphorylated SasS is able to transfer its phosphate to SasR. We propose that SasS and SasR form a two-component signal transduction system in the A signal transduction pathway. ^ To search for the genes regulated by SasS and SasR, expression patterns of a group of developmental genes were compared in wild-type and sasS null mutant backgrounds. SasS and SasR were found to positively regulate sasN and 4521. The sasN gene was previously identified as a negative regulator of 4521, located at about 170-bp downstream of sasR. It is required for normal fruiting body development. Based on the above data, a regulatory network consisting of sasS, sasR, sasN, and 4521 is hypothesized, and the interactions of the components in this network can now be further studied. ^

Dissecting Arabidopsis Gß signal transduction on the protein surface.

The heterotrimeric G-protein complex provides signal amplification and target specificity. The Arabidopsis (Arabidopsis thaliana) G?-subunit of this complex (AGB1) interacts with and modulates the activity of target cytoplasmic proteins. This specificity resides in the structure of the interface between AGB1 and its targets. Important surface residues of AGB1, which were deduced from a comparative evolutionary approach, were mutated to dissect AGB1-dependent physiological functions. Analysis of the capacity of these mutants to complement well-established phenotypes of G?-null mutants revealed AGB1 residues critical for specific AGB1-mediated biological processes, including growth architecture, pathogen resistance, stomata-mediated leaf-air gas exchange, and possibly photosynthesis. These findings provide promising new avenues to direct the finely tuned engineering of crop yield and traits.

Cytoplasmic RNA modulators of an inside-out signal-transduction cascade

A vaccinia virus-based RNA expression system enabled high-level cytoplasmic expression of RNA aptamers directed against the intracellular domain of the β2 integrin LFA-1, a transmembrane protein that mediates cell adhesion to intercellular adhesion molecule-1 (ICAM-1). In two different cell types, cytoplasmic expression of integrin-binding aptamers reduced inducible cell adhesion to ICAM-1. The aptamers specifically target, and thereby define, a functional cytoplasmic subdomain important for the regulation of cell adhesion in leukocytes. Our approach of aptamer-controlled blocking of signaling pathways in vivo could potentially be applied wherever targeted modulation of a signal-transduction cascade is desired.

The TOR (target of rapamycin) signal transduction pathway regulates the stability of translation initiation factor eIF4G in the yeast Saccharomyces cerevisiae

Initiation factor eIF4G is an essential protein required for initiation of mRNA translation via the 5′ cap-dependent pathway. It interacts with eIF4E (the mRNA 5′ cap-binding protein) and serves as an anchor for the assembly of further initiation factors. With treatment of Saccharomyces cerevisiae with rapamycin or with entry of cells into the diauxic phase, eIF4G is rapidly degraded, whereas initiation factors eIF4E and eIF4A remain stable. We propose that nutritional deprivation or interruption of the TOR signal transduction pathway induces eIF4G degradation.

ErbB2 expression increases the spectrum and potency of ligand-mediated signal transduction through ErbB4

Interleukin 3-dependent murine 32D cells do not detectably express members of the ErbB receptor family and do not proliferate in response to known ligands for these receptors. 32D transfectants were generated expressing human ErbB4 alone (32D.E4) or with ErbB2 (32D.E2/E4). Epidermal growth factor (EGF), neuregulin 1-β (NRG1-β), betacellulin (BTC), transforming growth factor-α (TGF-α), heparin binding-EGF (HB-EGF), and amphiregulin were analyzed for their ability to mediate mitogenesis in these transfectants. 32D.E4 responded mitogenically to NRG1-β and BTC. Surprisingly, EGF also induced significant DNA synthesis and TGF-α was negligibly mitogenic on 32D.E4 cells, whereas HB-EGF and amphiregulin were inactive. Although coexpression of ErbB2 with ErbB4 in 32D.E2/E4 cells did not significantly alter DNA synthesis in response to NRG1-β or BTC, it greatly enhanced mitogenesis elicited by EGF and TGF-α and unmasked the ability of HB-EGF to induce proliferation. EGF-related ligands that exhibited potent mitogenic activity on 32D.E2/E4 cells at low concentrations induced adherence, morphological alterations, and up-regulation of the Mac-1 integrin and FcγRII/III at higher concentrations. While 125I-EGF could be specifically crosslinked to both 32D.E4 and 32D.E2/E4 cells, its crosslinking capacity was greatly enhanced in the cotransfected cells. The ability of the various ligands to mediate proliferation and/or adhesion in the two transfectants correlated with their capacity to induce substrate tyrosine phosphorylation and to initiate and sustain activation of mitogen-activated protein kinase. We conclude that the ability of ErbB4 to mediate signal transduction through EGF-like ligands is broader than previously assumed and can be profoundly altered by the concomitant expression of ErbB2.

Interleukin-2 induces β2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

β2 integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of β2 integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4+ T cell lines obtained from healthy donors and a leukocyte adhesion deficiency (LAD) patient. We show that IL-2 induces tyrosine phosphorylation of a 125-kDa protein and homotypic adhesion in β2 integrin (CD18)-positive but not in β2-integrin-negative T cells. EDTA, an inhibitor of integrin adhesion, blocks IL-2-induced tyrosine phosphorylation of the 125-kDa protein but not other proteins in β2-integrin-positive T cells. Likewise, a β2 integrin (CD18) antibody selectively inhibits induction of the 125-kDa phosphotyrosine protein, whereas cytokine-mediated tyrosine phosphorylation of other proteins is largely unaffected. Immunoprecipitation experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in β2-integrin-positive but not in β2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fakB-tyrosine phosphorylation in β2-integrin-positive T cells. In parallel experiments, IL-2 does not induce or augment tyrosine phosphorylation of p125FAK. In conclusion, our data indicate that IL-2 induces β2-integrin-dependent signal transduction events involving the tyrosine kinase substrate fakB.

Platelet signal transduction defect with Gα subunit dysfunction and diminished Gαq in a patient with abnormal platelet responses

G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca2+ mobilization. Present studies demonstrated that platelet phospholipase A2 (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca2+ release induced by guanosine 5′-[γ-thio]triphosphate (GTP[γS]) was diminished in the patient’s platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of α-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A2 analog U46619. Binding of 35S-labeled GTP[γS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I2 analog) -induced rise in cAMP (mediated by Gαs) and its inhibition (mediated by Gαi) by thrombin in the patient’s platelet membranes were normal. Immunoblot analysis of Gα subunits in the patient’s platelet membranes showed a decrease in Gαq (<50%) but not Gαi, Gαz, Gα12, and Gα13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein α-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of Gαq in thrombin-induced responses.

Molecular switch in signal transduction: Reaction paths of the conformational changes in ras p21

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the γ-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD–Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of α2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of α2 and facilitates the unwinding of a helical turn of α2 (residues 66–69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.

Mechanical stretch induces vascular permeability factor in human mesangial cells: Mechanisms of signal transduction

Hemodynamic abnormalities have been implicated in the pathogenesis of the increased glomerular permeability to protein of diabetic and other glomerulopathies. Vascular permeability factor (VPF) is one of the most powerful promoters of vascular permeability. We studied the effect of stretch on VPF production by human mesangial cells and the intracellular signaling pathways involved. The application of mechanical stretch (elongation 10%) for 6 h induced a 2.4-fold increase over control in the VPF mRNA level (P < 0.05). There was a corresponding 3-fold increase in VPF protein level by 12 h (P < 0.001), returning to the baseline by 24 h. Stretch-induced VPF secretion was partially prevented both by the protein kinase C (PKC) inhibitor H7 (50 μM: 72% inhibition, P < 0.05) and by pretreatment with phorbol ester (phorbol-12-myristate-13 acetate 10−7 M: 77% inhibition, P < 0.05). A variety of protein tyrosine kinase (PTK) inhibitors, genistein (20 μg/ml), herbimycin A (3.4 μM), and a specific pp60src peptide inhibitor (21 μM) also significantly reduced, but did not entirely prevent, stretch-induced VPF protein secretion (respectively 63%, 80%, and 75% inhibition; P < 0.05 for all). The combination of both PKC and PTK inhibition completely abolished the VPF response to mechanical stretch (100% inhibition, P < 0.05). Stretch induces VPF gene expression and protein secretion in human mesangial cells via PKC- and PTK-dependent mechanisms.

Induction of host signal transduction pathways by Helicobacter pylori

Adherence of Helicobacter pylori to cultured gastric epithelial cells is associated with several cellular events, including the tyrosine phosphorylation of a 145-kDa host protein; the reorganization of the host cell actin and associated cellular proteins, like vasodilator-stimulated phosphoprotein, adjacent to the attached bacterial cell; and the subsequent release of the cytokine, interleukin 8 (IL-8). H. pylori isolated from patients with ulcer disease and gastric cancer contain a DNA insertion, the cag pathogenicity island (PAI), that is not present in bacteria isolated from individuals with asymptomatic infection. Mutations in a number of PAI genes abolish tyrosine phosphorylation and IL-8 synthesis but not the cytoskeletal rearrangements. Kinase inhibition studies suggest there are two distinct pathways operative in stimulating IL-8 release from host cells and one of these H. pylori pathways is independent of the tyrosine phosphorylation step.

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans

The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65PAK family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid “Spider” media. However, hyphal development was not impaired in some other media. A similar phenotype was caused by deletion of HST7, encoding a functional homolog of the S. cerevisiae Ste7p protein kinase. Overexpression of HST7 partially complemented the deletion of CST20. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans.