Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

THE ROLE OF RECEPTOR TYROSINE KINASE AXL IN PANCREATIC DUCTAL ADENOCARCINOMA AND ITS REGULATION BY HEMATOPOIETIC PROGENITOR KINASE 1

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive malignancies with less than 5% of five year survival rate. New molecular markers and new therapeutic targets are urgently needed for patients with PDA. Oncogenic receptor tyrosine kinase Axl has been reported to be overexpressed in many types of human malignancies, including diffuse glioma, melanoma, osteosarcoma, and carcinomas of lung, colon, prostate, breast, ovary, esophagus, stomach, and kidney. However, the expression and functions of Axl in PDA are unclear. We hypothesized that Axl contributes to the development and progression of PDA. We examined Axl expression in 54 human PDA samples and their paired benign pancreatic tissue by immunohistochemistry, we found that Axl was overexpressed in 70% of stage II PDAs, but only 22% of benign ducts (P=0.0001). Axl overexpression was associated with higher frequencies of distant metastasis and was an independent prognostic factor for both poor overall and recurrence-free survivals in patients with stage II PDA (p = 0.03 and 0.04). Axl silencing by shRNA in pancreatic cancer cell lines, panc-28 and Panc-1, decreased tumor cell migration and invasion and sensitized PDA cells to apoptosis stimuli such as γ-irradiation and serum starvation. In addition, we found that Axl-mediated Akt and NF-κB activation and up regulation of MMP2 were involved in the invasion, migration and survival of PDA cells. Thus, we demonstrate that Axl plays an important role in the development and progression of PDA. Targeting Axl signaling pathway may represent a new approach for the treatment of PDA. To understand the molecular mechanisms of Axl overexpression in PDA, we found that Axl expression was down-regulated by hematopoietic progenitor kinase 1 (HPK1), a newly identified tumor suppressor in PDA. HPK1 is lost in over 95% of PDAs. Restoration of HPK1 in PDA cells down-regulated Axl expression. HPK1-mediated Axl degradation was inhibited by leupeptin, baflomycin A1, and monensin, suggesting that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway. HPK1 interacted with and phosphorylated dynamin, a critical component of endocytosis pathway. Overexpression of dominant negative form of dynamin blocked the HPK1-mediated Axl degradation. Therefore we concluded that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway and loss of HPK1 expression may contribute to Axl overexpression in PDAs.

Transcriptional regulation of the {\it neu\/} oncogene and its negative regulation by the c-{\it myc\/} oncogene

The first part of my research involved the characterization of the neu gene promoter. I subcloned a 2.2-kb sequence located upstream to the extreme 5$\sp\prime$ end of the neu gene, in front of the bacterial reporter gene, chloramphenicol acetyltransferase (CAT). Transfection of this construct into different cell lines and subsequent CAT assays demonstrated that this 2.2-kb fragment was functional as a promoter. A series of deletion constructs was engineered to study the contribution of different fragments to transcription. Subcloning of individual fragments was followed by a cotransfection competition experiment, which demonstrated the involvement of protein factors interacting with the promoter. A gel retardation assay was also performed to show the physical binding of protein factors to the promoter. The combined results suggested that both positively and negatively acting protein factors are involved in interacting with different regions of the promoter, contributing to the overall transcription activity. My findings provide an insight into the regulation of neu gene expression, which in turn provides the tools to understand the molecular mechanisms of overexpression of the neu gene in some breast cancer and ovarian cancer cell lines.^ In the second part of my research, I discovered that another oncogene, c-myc, was able to reverse the transformed morphology that was induced by the neu oncogene. Utilizing the promoter constructs that I made, I was able to show that the c-myc oncogene has a negative regulatory effect on the expression of the neu oncogene. Further studies suggested that c-myc is able to lower the effective concentration of a positive factor(s) that interact with a 139-bp fragment of the neu gene promoter. These findings may provide a direct evidence of the long suspected role of the c-myc gene in transcriptional regulation. The neu gene may very well be the first identified mammalian target gene that is regulated by the c-myc oncogene. Since c-myc is known to be stimulated by various mitogenic signals and the neu gene is likely to be a growth factor receptor, it is possible that c-myc, when stimulated by the signal transduction pathway of the neu gene, would function as a negative feedback regulator on the neu gene receptor. (Abstract shortened with permission of author.) ^

The molecular mechanism of retinoic acid action: Regulation of tissue transglutaminase gene expression

Retinoic acid is a small lipophilic molecule that exerts profound effects on the growth and differentiation of both normal and transformed cells. It is also a natural morphogen that is critical in the development of embryonic structures. The molecular effects of retinoic acid involve alterations in the expression of several proteins and these changes are presumably mediated in part by alterations in gene expression. For instance, retinoic acid causes a rapid induction of tissue transglutaminase, an enzyme involved in protein cross-linking. The molecular mechanisms responsible for the effects of retinoic acid on gene expression have not been characterized. To approach this question, I have isolated and characterized tissue transglutaminase of cDNA clones. The deduced amino acid sequences of tissue transglutaminase and of factor XIIIa showed a relatively high degree of homology in their putative calcium binding domains.^ To explore the mechanism of induction of this enzyme, both primary (macrophages) and cultured cells (Swiss 3T3-C2 and CHO fibroblasts) were used. I found that retinoic acid is a general inducer of tissue transglutaminase mRNA in these cells. In murine peritoneal macrophages retinoic acid causes a rapid accumulation of this mRNA and this effect is independent of concurrent protein synthesis. The retinoic acid effect is not mediated by a post-transcriptional increase in the stability of the tissue transglutaminase mRNA, but appears to involve an increase in the transcription rate of the tissue transglutaminase gene. This provides the first example of regulation by retinoic acid of a specific gene, supporting the hypothesis that these molecules act by directly regulating the transcriptional activity of specific genes. A molecular model for the effects of retinoic acid on the expression of genes linked to cellular proliferation and differentiation is proposed. ^

Glutamate receptors expressed in {\it Xenopus\/} oocytes: Studies in function and regulation

The amino acid glutamate is the primary excitatory neurotransmitter for the CNS and is responsible for the majority of fast synaptic transmission. Glutamate receptors have been shown to be involved in multiple forms of synaptic plasticity such as LTP, LTD, and the formation of specific synaptic connections during development. In addition to contributing to the plasticity of the CNS, glutamate receptors also are involved in, at least in part, various pathological conditions such as epilepsy, ischemic damage due to stroke, and Huntington's chorea. The regulation of glutamate receptors, particularly the ionotropic NMDA and AMPA/KA receptors is therefore of great interest. In this body of work, glutamate receptor function and regulation by kinase activity was examined using the Xenopus oocyte which is a convenient and faithful expression system for exogenous proteins. Glutamate receptor responses were measured using the two-electrode voltage clamp technique in oocytes injected with rat total forebrain RNA. NMDA elicited currents that were glycine-dependent, subject to block by Mg$\sp{2+}$ in a voltage-dependent manner and sensitive to the specific NMDA antagonist APV in a manner consistent with those types of responses found in neural tissue. Similarly, KA-evoked currents were sensitive to the specific AMPA/KA antagonist CNQX and exhibited current voltage relationships consistent with the calcium permeable type II KA receptors found in the hippocampus. There is evidence to indicate that NMDA and AMPA/KA receptors are regulated by protein kinase A (PKA). We explored this by examining the effects of activators of PKA (forskolin, 1-isobutyl-3-methylxanthine (IBMX) and 8-Br-cAMP) on NMDA and KA currents in the oocyte. In buffer where Ca$\sp{2+}$ was replaced by 2 mM Ba$\sp{2+},$ forskolin plus IBMX and 8-Br-cAMP augmented currents due to NMDA application but not KA. This augmentation was abolished by pretreating the oocytes in the kinase inhibitor K252A. The use of chloride channel blockers resulted in attenuation of this effect indicating that Ba$\sp{2+}$ influx through the NMDA channel was activating the endogenous calcium-activated chloride current and that the cAMP mediated augmentation was at the level of the chloride channel and not the NMDA channel. This was confirmed by (1) the finding that 8-Br-cAMP increased chloride currents elicited via calcium channel activation while having no effect on the calcium channels themselves and (2) the fact that lowering the Ba$\sp{2+}$ concentration to 200 $\mu$M abolished the augmentation NMDA currents by 8-Br-cAMP. Thus PKA does not appear to modulate ionotropic glutamate receptors in our preparation. Another kinase also implicated in the regulation of NMDA receptors, calcium/phospholipid-dependent protein kinase (PKC), was examined for its effects on the NMDA receptor under low Ba$\sp{2+}$ (200 $\mu$M) conditions. Phorbol esters, activators of PKC, induced a robust potentiation of NMDA currents that was blockable by the kinase inhibitor K252A. Furthermore activation of metabotropic receptors by the selective agonist trans-ACPD, also potentiated NMDA albeit more modestly. These results indicate that neither NMDA nor KA-activated glutamate receptors are modulated by PKA in Xenopus oocytes whereas NMDA receptors appear to be augmented by PKC. Furthermore, the endogenous chloride current of the oocyte was found to be responsive to Ba$\sp{2+}$ and in addition is enhanced by PKA. Both of these latter findings are novel. In conclusion, the Xenopus oocyte is a useful expression system for the analysis of ligand-gated channel activity and the regulation of those channels by phosphorylation. ^

Regulation of two P450 isoforms: CYP2D6 and CYP4F11

Cytochromes P450 catalyze a monooxygenase reaction in which molecular oxygen is split and one oxygen atom is incorporated into the substrate. As a whole, P450 researchers have focused most of their attention on substrate metabolism and relatively little on how these enzymes are regulated. This study will focus on the regulation of two P450 isoforms known as, CYP2D6 and CYP4F11. ^ The human CYP2D gene locus contains two pseudogenes and one functional gene known as CYP2D6. This locus is highly polymorphic and produces several alternatively spliced transcripts from the pseudogene CYP2D7. My objective was to understand the role of SV5-in (splice variant 5), one of several alternative splice variants transcribed from the CYP2D7 pseudogene. My results indicate that SV5-in mRNA causes an increase in CYP2D6 protein levels and suggest that there is a role for SV5-in in regulation of CYP2D6 expression. ^ Second, CYP4F11 is a recently discovered and uncharacterized isoform, derived from the CYP4F subfamily. It metabolizes several clinically relevant drugs (i.e.—erythromycin and benzphetamine) and some endogenous inflammatory mediators (i.e.—LTB4). After evaluation of microarray data, I observed an increase in CYP4F11 mRNA levels from wild-type HCT116 cells compared to p53-null cells. Our objectives were to explore and understand this connection between p53 and CYP4F11. Microarray data were confirmed by Q-PCR, after which this effect was again observed at the protein level via Western blot and again at the promoter level via luciferase assay and chromatin immunoprecipitation. Our results indicate that p53 protein regulates expression of CYP4F11 mRNA and protein through CYP4F11 promoter binding (note that p53 binding to CYP4F11 DNA was not shown to be direct). These results signify a whole new level of regulation of drug metabolizing enzymes by p53. ^ An understanding of CYP4F11 regulation by p53 could help us understand another pathway leading to apoptosis or cell growth arrest. This can aid future drug studies and discover new drug metabolism pathways under the control of a tumor suppressor protein. An understanding of the CYP2D6 regulation pathway could illuminate the role of non-coding RNAs in the P450 field and potentially explain several inter-individual drug response variations observed in clinical medicine that are not yet completely explained by genotyping analysis. ^

Nonclassical mechanisms of progesterone action in the brain: I. Protein kinase C activation in the hypothalamus of female rats.

The modulation of gene regulation by progesterone (P) and its classical intracellular regulation by progestin receptors in the brain, resulting in alterations in physiology and behavior has been well studied. The mechanisms mediating the short latency effects of P are less well understood. Recent studies have revealed rapid nonclassical signaling action of P involving the activation of intracellular signaling pathways. We explored the involvement of protein kinase C (PKC) in P-induced rapid signaling in the ventromedial nucleus of the hypothalamus (VMN) and preoptic area (POA) of the rat brain. Both the Ca2+-independent (basal) PKC activity representing the activation of PKC by the in vivo treatments and the Ca+2-dependent (total) PKC activity assayed in the presence of exogenous cofactors in vitro were determined. A comparison of the two activities demonstrated the strength and temporal status of PKC regulation by steroid hormones in vivo. P treatment resulted in a rapid increase in basal PKC activity in the VMN but not the POA. Estradiol benzoate priming augmented P-initiated increase in PKC basal activity in both the VMN and POA. These increases were inhibited by intracerebroventricular administration of a PKC inhibitor administered 30 min prior to P. The total PKC activity remained unchanged demonstrating maximal PKC activation within 30 min in the VMN. In contrast, P regulation in the POA significantly attenuated total PKC activity +/- estradiol benzoate priming. These rapid changes in P-initiated PKC activity were not due to changes in PKC protein levels or phosphorylation status.

THE ROLE OF CYCLIN E IN PKC IOTA-DRIVEN EARLY OVARIAN TUMORIGENESIS

Among the gynecologic malignancies, epithelial ovarian tumors are the leading cause of death. For the past few decades, the only treatment has involved surgical resection of the tumor and/or general chemotherapies. In an attempt to improve treatment options, we have shown that several oncogenes that are overexpressed in ovarian cancer, PI3K, PKCiota, and cyclin E, all of which have been shown to lead to a poor prognosis and decreased survival, converge into a single pathway that could potentially be targeted therapeutically. Because of the ability of either PKCiota or cyclin E overexpression to independently induce anchorage-independent growth, a hallmark of cancer, we hypothesized that targeting PKCiota expression in ovarian cancer cells could induce a reversion of the transformed phenotype through down regulation of cyclin E. To test this hypothesis, we first established a correlation between PKCiota and cyclin E in a panel of 20 ovarian cancer cell lines. To show that PKCiota is upstream of cyclin E, PKCiota was stably knocked down using RNAi in IGROV cells (epithelial ovarian cancer cell line of serous histology). The silencing of PKCiota resulted in decreased expression of cell cycle drivers, such as cyclin D1/E and CDK2/4, and an increase in p27. These alteration in the regulators of the cell cycle resulted in a decrease in both proliferation and anchorage-independent growth, which was specifically through cyclin E, as determined by a rescue experiment. We also found that the mechanism of cyclin E regulation by PKCiota was at the level of degradation rather than transcription. Using two inhibitors to PI3K, we found that both the active form of PKCiota and total cyclin E levels decreased, implying that the PKCiota/cyclin E pathway is downstream from PI3K. In conclusion, we have identified a novel pathway in epithelial ovarian tumorigenesis (PI3K à PKCiota à Cyclin E à anchorage-independent growth), which could potentially be targeted therapeutically.

STUDY OF REST AS A NEGATIVE REGULATOR OF P16INK4A

STUDY OF REST AS A NEGATIVE REGULATOR OF P16INK4A Monica Gireud, B.S. Thesis Advisor: Vidya Gopalakrishnan, Ph.D. The RE1 Silencing Transcription Factor (REST) is a negative regulator of neuronal differentiation. It is expressed ubiquitously in early embryos, but downregulated in neural progenitors concomitant with onset of neuronal differentiation in these cells. REST has been widely studied as a negative regulator of neuronal differentiation genes. Our recent work identified a novel role for REST in control of cell proliferation. However, the underlying molecular mechanism(s) are not known and is a focus of the current thesis project. Here, we provide evidence that REST signaling controls the expression of the cyclin-dependent kinase inhibitor, p16Ink4a, a negative regulator of the cell cycle and passage through G1. We determined that REST expression in the proliferating granule progenitors of the cerebellum and its lack of expression in the differentiated neurons is reciprocally correlated with that of p16Ink4a. Decline in REST levels in differentiating primary and neural stem cells immortalized with v-myc (NSC-M) granule progenitors in vitro was also associated with upregulation of p16Ink4a expression. Conversely, constitutive human REST transgene expression in NSC-M cells (NSC-MRs) blocked p16Ink4 upregulation, even under neuronal differentiation conditions. However, the lack of a consensus REST DNA binding RE1 element in the regulatory regions of p16Ink4a locus suggested an indirect regulation of p16Ink4a by REST. Based on work from other groups that showed repression of p16Ink4a transcription by the polycomb protein Bmi-1, and its negative regulation by microRNA-203 (miR-203) and our identification of a RE1 element in the downstream regulatory region of miR-203, we asked if the p16Ink4a expression was controlled by REST through a series of negative regulatory events involving miR-203 and Bmi-1. We observed that Bmi1 -expression mirrored that of REST and inversely correlated with that of miR-203 in the postnatal cerebellum and in vitro differentiated granule and NSC-M progenitors. In contrast, forced REST transgene expression in NSC-MR cells abrogated the decrease in Bmi-1 levels and elevation in miR-203 expression. Significant REST binding to the miR-203 RE1 element was also observed in NSC-M cells, indicating that REST had the potential to directly regulate miR-203 expression. In conclusion, our studies suggest a role for REST in control of cell cycle transit in neural progenitors through negative regulation of p16Ink4a. Further validation of these results in REST knockout mice is needed, and is ongoing.

Functional characterization of transcription factor USF

USF, Upstream Stimulatory Factor, is a family of ubiquitous transcription factors that contain highly conserved basic helix-loop-helix leucine zipper DNA binding domains and recognize the core DNA sequence CACGTG. In human and mouse, two members of the USF family, USF1 and USF2, encoded by two different genes, contribute to the USF activity. In order to gain insights into the mechanisms by which USFs function as transcriptional activators, different approaches were used to map the domains of USF2 responsible for nuclear localization and transcriptional activation. Two stretches of amino acids, one in the basic region of the DNA binding domain, the other in a highly conserved N-terminal region, were found to direct nuclear localization independently of one another. Two distinct activation domains were also identified. The first one, located in the conserved N-terminal region that overlaps the C-terminal nuclear localization signal, functioned only in the presence of an initiator element in the promoter of the reporter. The second, in a nonconserved region, activated transcription in the absence of an initiator element or when fused to a heterologous DNA binding domain. These results suggest that USF2 functions in different promoter contexts by selectively utilizing different activation domains.^ The deletion analysis of USF2 also identified two dominant negative mutants of USF, one lacking the activation domain, the other lacking the basic domain. The latter proved useful for testing the direct involvement of USFs in the transcriptional activation mediated by the viral protein IE62.^ To investigate the biological function of USFs, foci and colony formation assays were used to study the growth regulation by USFs. It was found that USFs had a strong antagonistic effect on cellular transformation mediated by the bHLH/LZ protein Myc. This effect required the DNA binding activity of either USF 1 or USF2. Moreover, USF2, but not USF1 or other mutants of USFs, was also found to have strong inhibitory effect on the cellular transformation by E1a and on the growth of HeLa cells. These results demonstrate that USFs could potentially regulate growth through two mechanisms, one by antagonizing the function of Myc in cellular transformation, the other by mediating a more general growth inhibitory effect. ^

STUDY OF THE EFFECTS OF TOTAL MODULATION TRANSFER FUNCTION CHANGES ON OBSERVER PERFORMANCE USING CLINICAL MAMMOGRAMS

The main goal of this study was to relate physical changes in image quality measured by Modulation Transfer Function (MTF) to diagnostic accuracy.^ One Hundred and Fifty Kodak Min-R screen/film combination conventional craniocaudal mammograms obtained with the Pfizer Microfocus Mammographic system were selected from the files of the Department of Radiology, at M.D. Anderson Hospital and Tumor Institute.^ The mammograms included 88 cases with a variety of benign diagnosis and 62 cases with a variety of malignant biopsy diagnosis. The average age of the patient population was 55 years old. 70 cases presented calcifications with 30 cases having calcifications smaller than 0.5mm. 46 cases presented irregular bordered masses larger than 1 cm. 30 cases presented smooth bordered masses with 20 larger than 1 cm.^ Four separated copies of the original images were made each having a different change in the MTF using a defocusing technique whereby copies of the original were obtained by light exposure through different thicknesses (spacing) of transparent film base.^ The mammograms were randomized, and evaluated by three experienced mammographers for the degree of visibility of various anatomical breast structures and pathological lesions (masses and calicifications), subjective image quality, and mammographic interpretation.^ 3,000 separate evaluations were anayzed by several statistical techniques including Receiver Operating Characteristic curve analysis, McNemar test for differences between proportions and the Landis et al. method of agreement weighted kappa for ordinal categorical data.^ Results from the statistical analysis show: (1) There were no statistical significant differences in the diagnostic accuracy of the observers when diagnosing from mammograms with the same MTF. (2) There were no statistically significant differences in diagnostic accuracy for each observer when diagnosing from mammograms with the different MTF's used in the study. (3) There statistical significant differences in detail visibility between the copies and the originals. Detail visibility was better in the originals. (4) Feature interpretations were not significantly different between the originals and the copies. (5) Perception of image quality did not affect image interpretation.^ Continuation and improvement of this research ca be accomplished by: using a case population more sensitive to MTF changes, i.e., asymptomatic women with minimum breast cancer, more observers (including less experienced radiologists and experienced technologists) must collaborate in the study, and using a minimum of 200 benign and 200 malignant cases.^

Novel mechanisms for PKC family enzymes to regulate PI3K signaling pathway

Phosphatidylinositol 3-kinase (PI3K) generates membrane phospholipids that serve as second messengers to recruit signaling proteins to plasma membrane consequently regulating cell growth and survival. PI3K is a heterodimer consisting of a catalytic p110 subunit and a regulatory p85 subunit. Association of the p85 with other signal proteins is critical for induced PI3K activation. Activated PI3K, in turn, leads to signal flows through a variety of PI3K effectors including PDK1, AKT, GSK3, BAD, p70 S6K and NFκB. The PI3K pathway is under regulation by multiple signal proteins representing cross-talk between different signaling cascades. In this study, we have evaluated the role of protein kinase C family kinases on signaling through PI3K at multiple levels. Firstly, we observed that the action of PKC specific inhibitors like Ro-31-8220 and GF109203X was associated with an increased AKT phosphorylation and activity, suggesting that PKC kinases might play a negative role in the regulation of PI3K pathway. Then, we demonstrated the stimulation of AKT by PKC inhibition was dependent on functional PI3K enzyme and able to be transmitted to the AKT effector p70 S6K. Furthermore, we showed an inducible physical association between the PKCζ isotype and AKT, which was accompanied by an attenuated AKT activity. However, a kinase-dead form of PKC failed to affect AKT. In the second part of our research we revealed the ability of a different PKC family member, PKCδ to bind to the p85 subunit of PI3K in response to oxidative stress, a process requiring the activity of src tyrosine kinases. The interaction was demonstrated to be a direct and specific contact between the carboxyl terminal SH2 domain of p85 and tyrosine phosphorylated PKCδ. Several different types of agonists were capable to induce this association including tyrosine kinases and phorbol esters with PKCδ tyrosine phosphorylation being integral components. Finally, the PKCδ-PI3K complex was related to a reduction in the AKT phosphorylation induced by src. A kinase-deficient mutant of PKCδ was equally able to inhibit AKT signal as the wild type, indicative of a process independent of PKCδ catalytic activity. Altogether, our data illustrate different PKC isoforms regulating PI3K pathway at multiple levels, suggesting a mechanism to control signal flows through PI3K for normal cell activities. Although further investigation is required for full understanding of the regulatory mechanism, we propose that complex formation of signal proteins in PI3K pathway and specific PKC isoforms plays important role in their functional linkage. ^

Mechanisms of heterosynaptic modulation of transmission in Aplysia

Heterosynaptic plasticity has received considerable attention as a means to induce and maintain cell-wide, as opposed to synapse-specific, learning-related modifications. Modulatory neurotransmitters are thought to provide the attentional and motivational state for memory formation. However, the cellular and molecular mechanisms mediating the effects of most of these modulators on synaptic plasticity and learning remain unclear. A well established system for the study of heterosynaptic plasticity is the Aplysia sensorimotor synapse, which is subject regulation by at least two neuromodulators, serotonin (5-HT) and FMRFa. ^ 5-HT engages multiple second messenger cascades to induce short- and long-term facilitation (STF and LTF, respectively) of synaptic transmission. One mechanism proposed to be involved in STF is mobilization of synaptic vesicles from a storage pool to a releasable pool. To investigate this hypothesis, we examined the involvement of the protein synapsin, a central element in the regulation of the storage pool of vesicles in nerve terminals, in STF. 5-HT induced phosphorylation of synapsin and modified its subcellular distribution via PKA and p42/44 MAPK. Electrophysiological experiments and computer simulations suggested that synapsin can support heterosynaptic plasticity by regulating vesicle mobilization. ^ FMRFa induce short- and long-term synaptic depression in Aplysia . Long-term depression (LTD) correlates with morphological changes, the mechanisms of which remain elusive. LTD is also transcription- and translation-dependent, but little is known about the genes expressed and their regulation. We investigated the role of protein degradation via the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by the transcription factor CREB2, which is generally regarded as a transcription repressor. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions. ^ These and additional studies on the interaction of the 5-HT and FMRFa-activated pathways suggest that different neuromodulators, by activating several and sometimes overlapping signaling cascades, can exercise bidirectional control on synaptic gain and information processing.^

NHERF1 recruits the tumor suppressors PTEN and PHLPP to synergistically inhibit the PI-3K pathway

Glioblastoma multiforme is the most common form of brain cancer that presents patients with a poor prognosis that has remained unchanged over the past few decades. The tumor suppressor phosphatase PTEN antagonizes one of the major oncogenic pathways involved in the progression of glioblastoma, and is frequently deleted in this cancer type. Contrary to our expectations, we found that most glioblastoma cells expressing endogenous PTEN also harbor basal PI-3K/AKT activation mainly attributable to impaired PTEN membrane localization. This alteration correlated with a shift of the adaptor protein NHERF1, which contributes to PTEN membrane recruitment in normal cells, from the membrane to the cytoplasm. In cells expressing membrane-localized NHERF1, only simultaneous PTEN and NHERF1 depletion achieved AKT activation, suggesting the involvement of additional PI-3K/AKT suppressor regulated by NHERF1. We identified these novel interactors of NHERF1 as the PHLPP1 and PHLPP2 phosphatases. ^ NHERF1 directly interacted and recruited both PHLPP proteins to the membrane and, through both NHERF1 PDZ domains, assembled ternary complexes consisting of PTEN-NHERF1-PHLPP. Only simultaneous depletion of PTEN and PHLPP1 significantly activated AKT and increased proliferation in cells with membrane-localized NHERF1. Analysis of glioblastoma human tumors revealed frequent loss of membrane-localized NHERF1. On the other hand, targeting of NHERF1 to the membrane achieved suppression of AKT and cell proliferation. Our findings reveal a novel mechanism for PI-3K/AKT regulation by the synergistic cooperation between two important tumor suppressors, PTEN and PHLPP, via the scaffold protein NHERF1. ^