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.

EXPRESSION AND IMMUNOLOGICAL CHARACTERIZATION OF HERV-K TRANSMEMBRANE ENVELOPE PROTEIN IN HUMAN BREAST CANCER

The human endogenous retrovirus K (HERV-K) env gene encodes envelope protein comprising surface (SU) and transmembrane (TM) domains. Having shown the exclusive expression of SU in human breast cancer and the stimulation of SU-specific immune responses in patients with breast cancer, our research here confirmed and extended the data by investigating the expression of HERV-K TM envelope domain and the induction of specific immune responses against TM in breast cancer patients. We found HERV-K TM mRNA and protein expression only in human breast cancer cells but not in normal controls. The specific immune responses against TM domain were induced in mice determined by enzyme-linked immunosorbent assay (ELISA) and IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay. Furthermore, ELISA detected higher titers of anti-HERV-K TM Env IgG antibodies in sera of breast cancer patients. In addition, the magnitude of the anti-HERV TM B cell response was correlated with the disease stage. Peripheral blood mononuclear cells (PBMCs) before and after in vitro stimulation (IVS) with HERV-K TM from patients with breast cancer as well as healthy controls were tested for T cell responses against HERV-K TM domain by ELISPOT assay. Breast cancer patients (n=21) had stronger HERV-K TM-specific cellular responses than healthy controls (n=12) (P < 0.05). These findings suggest, for the first time, that HERV-K TM expression was enhanced in human breast cancer cells and was able to induce specific B cell and T cell immune responses in breast cancer patients. This study provides support for HERV-K TM as a promising source of antigen for anti-tumor immunotherapy, prevention, diagnosis, and prognosis.

Multiple site phosphorylation of tyrosine hydroxylase: A potential role for protein kinase C in the regulation of catecholamine synthesis

Tyrosine hydroxylase (TH), the initial and rate limiting enzyme in the catecholaminergic biosynthetic pathway, is phosphorylated on multiple serine residues by multiple protein kinases. Although it has been demonstrated that many protein kinases are capable of phosphorylating and activating TH in vitro, it is less clear which protein kinases participate in the physiological regulation of catecholamine synthesis in situ. These studies were designed to determine if protein kinase C (PK-C) plays such a regulatory role.^ Stimulation of intact bovine adrenal chromaffin cells with phorbol esters results in stimulation of catecholamine synthesis, tyrosine hydroxylase phosphorylation and activation. These responses are both time and concentration dependent, and are specific for those phorbol ester analogues which activate PK-C. RP-HPLC analysis of TH tryptic phosphopeptides indicate that PK-C phosphorylates TH on three putative sites. One of these (pepetide 6) is the same as that phosphorylated by both cAMP-dependent protein kinase (PK-A) and calcium/calmodulin-dependent protein kinase (CaM-K). However, two of these sites (peptides 4 and 7) are unique, and, to date, have not been shown to be phosphorylated by any other protein kinase. These peptides correspond to those which are phosphorylated with a slow time course in response to stimulation of chromaffin cells with the natural agonist acetylcholine. The activation of TH produced by PK-C is most closely correlated with the phosphorylation of peptide 6. But, as evident from pH profiles of tyrosine hydroxylase activity, phosphorylation of peptides 4 and 7 affect the expression of the activation produced by phosphorylation of peptide 6.^ These data support a role for PK-C in the control of TH activity, and suggest a two stage model for the physiological regulation of catecholamine synthesis by phosphorylation in response to cholinergic stimulation. An initial fast response, which appears to be mediated by CaM-K, and a slower, sustained response which appears to be mediated by PK-C. In addition, the multiple site phosphorylation of TH provides a mechanism whereby the regulation of catecholamine synthesis appears to be under the control of multiple protein kinases, and allows for the convergence of multiple, diverse physiological and biochemical signals. ^

Molecular signals in B lymphocyte activation: The role of intracellular calcium ion concentration, protein kinase C activation, and autocrine interleukin-2

Calcium ionophore, ionomycin, and phorbol myristate acetate (PMA) were used to activate rabbit peripheral blood B cells to study the role of increased intracellular calcium ion concentration ( (Ca$\sp2+\rbrack\sb{\rm i}$), protein kinase C (PKC) activation, and autocrine interleukin (IL-2) in inducing cell cycle entry and maintaining activation to DNA synthesis. When stimulated with a combination of ionomycin and PMA the B cells produced a soluble factor that supported the IL-2 dependent cell line, CTLL-2. The identity of the factor was established as IL-2 and its source was proved to be B cells in further experiments. Absorption studies and limiting dilution analysis indicated that IL-2 produced by B cells can act as an autocrine growth factor. Next, the effect of complete and incomplete signalling on B lymphocyte activation leading to cell cycle entry, IL-2 production, functional IL-2 receptor (IL-2R) expression, and DNA synthesis was examined. It was observed that cell cycle entry could be induced by signals provided by each reagent alone, but IL-2 production, IL-2R expression, and progression to DNA synthesis required activation with both reagents. Incomplete activation with ionomycin or PMA alone altered the responsiveness of B cells to further stimulation only in the case of ionomycin, and the unresponsiveness of these cells was apparently due to a lack of functional IL-2R expression on these cells, even though IL-2 production was maintained. The requirement of IL-2 for maintenance of activation to DNA synthesis was then investigated. The hypothesis that IL-2, acts in late G$\sb1$ and is required for DNA synthesis in B cells was supported by comparing IL-2 production and DNA synthesis in peripheral blood cells and purified B cells, kinetic analysis of these events in B cells, effects of anti-IL-2 antibody and PKC inhibitors, and by the response of G$\sb1$ B cells. Additional signals transduced by the interaction of autocrine IL-2 and functional IL-2 receptor on rabbit B cells were found to be necessary to drive these cells to S phase, after initial activation caused by simultaneous increase in (Ca$\sp2+\rbrack\sb{\rm i}$ and PKC activation had induced cell cycle entry, IL-2 production, and functional IL-2 receptor expression. ^

Mechanisms of protein regulation in rat skeletal muscle during resistance exercise and training

The cellular mechanisms through which adult rat skeletal muscle protein is regulated during resistance exercise and training was investigated. A model of non-voluntary resistance exercise was described which involves the electrically-stimulated contraction of the lower leg muscles of anesthetized rats against a weighted pulley-bar. Muscle protein synthesis rates were measured by in vivo constant infusion of $\sp3$H-leucine following a single bout of resistance exercise. Specific messenger RNA levels were determined by dot-blot hybridization analysis using $\sp{32}$P-labelled DNA probes after a single bout and multiple bouts of phasic training. The effects of phasic training on increasing skeletal muscle mass was assessed. Between 12 and 36 hours following a single resistance exercise bout (24-192 contractions), total mixed and myofibril protein synthesis rates were significantly increase (32%-65%) after concentric (gastrocnemius m.) and eccentric (tibialis anterior m.) contractions. Eccentric contractions had greater effects on myofibril synthesis with more prolonged increases in synthesis rates. Lower numbers of eccentric than concentric contractions were required to increase synthesis. Cellular RNA was increased after exercise but the relative levels of skeletal $\alpha$-actin and cytochrome c mRNAs were unchanged. Since increases in synthesis rates exceeded increases in RNA, post-transcriptional mechanisms may be primarily responsible for increased protein synthesis after a resistance exercise bout. After 10-22 weeks of phasic eccentric resistance training, muscle enlargement (16%-30%) was produced in the tibialis anterior m. after all training paradigms examined. In contrast, gastrocnemius m. enlargement after phasic concentric training occurred after moderate (24/bout) but not after high (192/bout) repetition training. The absence of muscle growth in the gastrocnemius m. after high repetition training despite increased synthesis rates after the initial bout and RNA and possibly mRNA accumulation during training suggests a role for post-translational mechanisms (protein degradation) in the control of muscle growth in the gastrocnemius m. It is concluded that muscle protein during resistance exercise and training is regulated at several cellular levels. The particular response may be influenced by the exercise intensity and duration, the training frequency and the type of contractile work (eccentric vs. concentric) performed. ^

cAMP-dependent protein kinase and heterologous desensitization of the adenylyl cyclase

Previous experiments had shown no differences in desensitization in cells with mutations of the adenylyl cyclase or the cAMP-dependent protein kinase and had ruled out this kinase as a mediator of desensitization; however, the assays of adenylyl cyclase had been made at high concentrations of free magnesium. The work presented in this dissertation documents a role for cAMP-dependent protein kinase which became apparent with assays at low concentrations of free magnesium. (1) The adenylyl cyclase in membranes from wild type S49 lymphoma cells showed substantial desensitization after incubation of the intact cells with low concentrations of epinephrine (5-20 nM). This desensitization was heterologous, that is it reduced the subsequent responses of the adenylyl cyclase to both epinephrine and prostaglandin-E$\sb1$. (2) The adenylyl cyclase in membranes of S49 cyc$\sp-$ cells, which do not make cAMP in response to hormones, and S49 kin$\sp-$ cells, which lack cAMP-dependent protein kinase activity, showed no heterologous desensitization following incubation of the intact cells with low concentrations of hormones. (3) Heterologous desensitization of the adenylyl cyclase was induced by incubations of wild type cells with forskolin, which activates the adenylyl cyclase downstream of the hormone receptors, or dibutyryl-cAMP, which activates the cAMP-dependent protein kinase directly. (4) Site-directed mutagenesis was used to delete the cAMP-dependent protein kinase consensus phosphorylation sequences on the $\beta$-adrenergic receptor. Heterologous desensitization occurred in intact L-cells expressing the wild type receptor or the receptor lacking the C-terminal phosphorylation site; however, only homologous desensitization occurred when the phosphorylation site on the third intracellular loop of the receptor was deleted. (5) To test directly the effects of cAMP-dependent protein kinase on the adenylyl cyclase the catalytic subunit of the kinase was purified from bovine heart and incubated with adenylyl cyclase in plasma membrane preparations. In this cell-free system the kinase caused rapid heterlogous reductions of the responsiveness of the S49 wild type adenylyl cyclase. Additionally, the adenylyl cyclase in kin$\sp-$ membranes, which showed only homologous desensitization in the intact cell, was desensitization by cell-free incubation with the kinase.^ The epinephrine responsiveness was not affected in L-cell membranes expressing the $\beta$-adrenergic receptor lacking the cAMP-dependent protein kinase consensus sequence on the third intracellular loop. ^

Fatty acylated proteins and the protein kinase C signaling pathway

Numerous proteins in intracellular signaling pathways are known to be covalently modified by long chain fatty acids. The objective of this project was to identify potentially novel components of the protein kinase C signaling pathway by virtue of their fatty acylation. A 64 kDa palmitoylated protein (p64) was identified that became deacylated following stimulation of quiescent cells with serum, FGF, or PDBu, suggesting that stimulus-dependent deacylation might alter interactions between p64 and other membrane/cytoskeletal components. A myristoylated protein of 68 kDa observed during these studies was identified as the "80K" PKC substrate. This protein was acylated cotranslationally with myristate through an amide linkage. The majority of the 80K protein was tightly associated with the plasma membrane, with approximately 20% in the cytosol. Although phosphorylation of the membrane-bound and soluble forms of the protein was increased 6-fold in response to PDBu, no changes in the subcellular distribution or myristoylation of the protein were observed. A cDNA encoding the murine form of this protein was cloned, and its deduced amino acid sequence revealed the presence of an N-terminal myristoylation consensus and five potential sites for phosphorylation by PKC. A mutant in which the N-terminal glycine residue was changed to alanine was no longer a substrate for NMT and consequently lost its membrane-binding potential. However, its ability to be phosphorylated in response to purified growth factors and phorbol esters was unimpaired. These results indicate that the myristoylated N-terminus of the 80K protein is required for its association with the plasma membrane, and that the cytoplasmic form of the protein can be phosphorylated independently of the membrane-bound form. Mutants of PKC were constructed in which the regulatory domain was removed and replaced by the N-terminus of the 80K or Al proteins. Unexpectedly, both the myristoylated and nonmyristoylated fusion proteins were tightly associated with the nuclear envelope. Further deletion analyses mapped nuclear targeting signals to the hinge region and a portion of the catalytic domain of PKC, explaining the ability of PKC to be translocated to the nucleus in response to certain stimuli. ^

Analyses of pp60$\sp{{\rm c}-src}$ and epidermal growth factor receptor protein tyrosine kinases in a human colon adenocarcinoma cell line following induction of goblet cell-like characteristics by tumor necrosis factor-alpha

Regulation of colonic epithelial cell proliferation and differentiation remains poorly understood due to the inability to design a model system which recapitulates these processes. Currently, properties of "differentiation" are studied in colon adenocarcinoma cell lines which can be induced to express some, but not all of the phenotypes of normal cells. In this thesis, the DiFi human colon adenocarcinoma cell line is utilized as an in vitro model system in which to study mucin production. In response to treatment with tumor necrosis factor-alpha, DiFi cells acquire some properties of mucin-producing goblet cells including altered morphology, increased reactivity to wheat germ agglutinin, and increased mucin production as determined by RNA expression as well as reactivity with the MUC-1 antibodies, HMFG-1 and SM-3. Thus, TNF-treated DiFi cells represent one of the few in vitro systems in which mucin expression can be induced.^ DiFi cells express an activated pp60$\sp{{\rm c}-src},$ as do most colon adenocarcinomas and derived cell lines, as well as an amplified epidermal growth factor (EGF) receptor. To assess potential changes in these enzymes during induction of differentiation characteristics, potential changes in the levels and activities of these enzymes were examined. For pp60$\sp{{\rm c}-src},$ no changes were observed in protein levels, specific activity of the kinase, cellular localization, or phosphorylation pattern as determined by Staphylococcus aureus V8 protease partial proteolytic mapping after induction of goblet cell-like phenotypic changes. These results suggest that pp60$\sp{{\rm c}-src}$ is regulated differentially in goblet cells than in absorptive cells, as down-modulation of pp60$\sp{{\rm c}-src}$ kinase occurs in the latter. Therefore, effects on pp60$\sp{{\rm c}-src}$ may be critical in colon regulation, and may be important in generating the various colonic epithelial cell types.^ In contrast to pp60$\sp{{\rm c}-src},$ EGF receptor tyrosine kinase activity decreased ($<$5-fold) after TNF treatment and at the time in which morphologic changes were observed. Similar decreases in tyrosine phosphorylation of EGF receptor were observed as assessed by immunoblotting with an anti-phosphotyrosine antibody. In addition, ($\sp{125}$I) -EGF cell surface binding was reduced approximately 3-fold following TNF treatment with a concomitant reduction in receptor affinity ($<$2-fold). These results suggest that modulation of EGF receptor may be important in goblet cell differentiation. In contrast, other published studies have demonstrated that increases in EGF receptor mRNA and in ($\sp{125}$I) -EGF binding accompany differentiation toward the absorptive cell phenotype. Therefore, differential regulation of both EGF receptor and pp60$\sp{{\rm c}-src}$ occur along the goblet cell and absorptive cell differentiation pathways. Thus, my results suggest that TNF-treated DiFi cells represent a unique system in which to study distinct patterns of regulation of pp60$\sp{{\rm c}-src}$ and EGF receptor in colonic cells, and to determine if increased MUC-1 expression is an early event in goblet cell differentiation. ^

Transcriptional analysis of liver-specific expression and differential interleukin-6 response of rat kininogen genes

Analyses of rat T1 kininogen gene/chloramphenicol acetyltransferase (T1K/CAT) constructs revealed two regions important for tissue-specific and induced regulation of T1 kininogen.^ Although the T1 kininogen gene is inducible by inflammatory cytokines, a highly homologous K kininogen gene is minimally responsive. Moreover, the basal expression of a KK/CAT construct was 5- to 7-fold higher than that of the analogous T1K/CAT construct. To examine the molecular basis of this differential regulation, a series of promoter swapping experiments was carried out. Our transfection results showed that at least two regions in the K kininogen gene are important for its high basal expression: a distal 19-bp region (C box) constituted a binding site for CCAAT/enhancer binding protein (C/EBP) family proteins and a proximal 66-bp region contained two adjacent binding sites for hepatocyte nuclear factor-3 (HNF-3). The distal HNF-3 binding site from the K kininogen promoter demonstrated a stronger affinity than that from the T1 kininogen promoter. Since C/EBP and HNF-3 are highly enriched in the liver and known to enhance transcription of liver-specific genes, differential binding affinities of these factors accounted for the higher basal expression of the K kininogen gene.^ In contrast to the K kininogen C box, the T1 kininogen C box does not bind C/EBP presumably due to their two-nucleotide divergence. This sequence divergence, however, converts it to a consensus binding sequence for two IL-6-inducible transcription factors--IL-6 response element binding protein and acute-phase response factor. To functionally determine whether C box sequences are important for their differential acute-phase response, T1 and K kininogen C boxes were swapped and analyzed after transfection into Hep3B cells. Our results showed that the T1 kininogen C box is indeed one of the IL-6 response elements in T1 kininogen promoter. Furthermore, its function can be modulated by a 5$\sp\prime$-adjacent C/EBP-binding site (B box) whose mutation significantly reduced the overall induced activity. Moreover, this B box is the target site for binding and transactivation of another IL-6 inducible transcription factor C/EBP$\delta.$ Evolutionary divergence of a few critical nucleotides can either lead to subtle changes in the binding affinities of a given transcription factor or convert a binding sequence for a constitutive factor to a site recognized by an inducible factor. (Abstract shortened by UMI.) ^

Effects of variable Gs expression on $\beta\sb2$-adrenergic receptor stimulated adenylyl cyclase response

An exact knowledge of the kinetic nature of the interaction between the stimulatory G protein (G$\sb{\rm s}$) and the adenylyl cyclase catalytic unit (C) is essential for interpreting the effects of Gs mutations and expression levels on cellular response to a wide variety of hormones, drugs, and neurotransmitters. In particular, insight as to the association of these proteins could lead to progress in tumor biology where single spontaneous mutations in G proteins have been associated with the formation of tumors (118). The question this work attempts to answer is whether the adenylyl cyclase activation by epinephrine stimulated $\beta\sb2$-adrenergic receptors occurs via G$\sb{\rm s}$ proteins by a G$\sb{\rm s}$ to C shuttle or G$\sb{\rm s}$-C precoupled mechanism. The two forms of activation are distinguishable by the effect of G$\sb{\rm s}$ levels on epinephrine stimulated EC50 values for cyclase activation.^ We have made stable transfectants of S49 cyc$\sp-$ cells with the gene for the $\alpha$ protein of G$\sb{\rm s}$ $(\alpha\sb{\rm s})$ which is under the control of the mouse mammary tumor virus LTR promoter (110). Expression of G$\sb{\rm s}\alpha$ was then controlled by incubation of the cells for various times with 5 $\mu$M dexamethasone. Expression of G$\sb{\rm s}\alpha$ led to the appearance of GTP shifts in the competitive binding of epinephrine with $\sp{125}$ICYP to the $\beta$-adrenergic receptors and to agonist dependent adenylyl cyclase activity. High expression of G$\sb{\rm s}\alpha$ resulted in lower EC50's for the adenylyl cyclase activity in response to epinephrine than did low expression. By kinetic modelling, this result is consistent with the existence of a shuttle mechanism for adenylyl cyclase activation by hormones.^ One item of concern that remains to be addressed is the extent to which activation of adenylyl cyclase occurs by a "pure" shuttle mechanism. Kinetic and biochemical experiments by other investigators have revealed that adenylyl cyclase activation, by hormones, may occur via a Gs-C precoupled mechanism (80, 94, 97). Activation of adenylyl cyclase, therefore, probably does not occur by either a pure "'Shuttle" or "Gs-C Precoupled" mechanism, but rather by a "Hybrid" mechanism. The extent to which either the shuttle or precoupled mechanism contributes to hormone stimulated adenylyl cyclase activity is the subject of on-going research. ^

Identification of two vitamin D response elements in the rat osteopontin gene

Studies to elucidate the function of vitamin D have demonstrated an important role in regulating bone-related cells, including osteoblasts and osteoclasts. A seemingly paradoxical observation is that 1,25(OH)$\sb2$D$\sb3$, the active metabolite of vitamin D, stimulates bone resorption, yet regulates transcription of genes expressed by osteoblasts. One mechanism that could explain these actions is the upregulation of transcription of osteoblast-specific genes. These gene products could then act as effectors to influence osteoclastic activity. We hypothesized that molecular signals could be deposited directly into the mineralized matrix in the form of noncollagenous proteins, such as osteopontin (OPN). The structure, biosynthesis and localization of OPN suggest that it could function to mediate the molecular "cross talk" between osteoblasts and osteoclasts in response to 1,25(OH)$\sb2$D$\sb3$. To begin to address this hypothesis, elucidation of the molecular mechanisms of action involved in the transactivation of OPN by 1,25(OH)$\sb2$D$\sb3$ is essential.^ In the present study, the rat opn gene was isolated and characterized. Functional analysis by transient transfection of the 5$\sp\prime$ flanking sequences of the rat opn gene fused to the luciferase gene demonstrated that OPN is transcriptionally upregulated by 1,25(OH)$\sb2$D$\sb3$, mediated through two vitamin D response elements (VDRE). Both proximal and distal VDREs are structurally similar (two imperfect direct repeats separated by a 3 nucleotide spacer) and bind protein complexes that include the VDR and retinoid-X receptor (RXR). Isolated VDRE expression constructs produce functional activity of equivalent magnitude of responsiveness to 1,25(OH)$\sb2$D$\sb3$. However, expression constructs containing either VDRE and at least 200 bp of 5$\sp\prime$ and 3$\sp\prime$ flanking sequence demonstrated that the distal VDRE produces an amplitude of response significantly higher than the proximal VDRE. We conclude that the transcriptional upregulation of the opn gene by 1,25(OH)$\sb2$D$\sb3$ involves the transactivation of two VDREs, while maximal responsiveness requires interaction of the VDREs with additional cis-elements contained in the 5$\sp\prime$ sequence. ^

Biochemical characterization of PICK1, a PKC binding protein

Protein kinase C (PKC) is a family of serine-threonine kinases that are activated by a wide variety of hormones, neurotransmitters and growth factors. A single cell type contains multiple isoforms that are translocated to distinct and different subcellular sites upon mitogenic stimulus. Many different cellular responses are attributed to PKC activity though relatively few substrates or binding proteins have been definitively characterized. We used the hinge and catalytic domain of PKC$\alpha$ (PKC7) in a yeast two-hybrid screen to clone proteins that interact with C-kinase (PICKs). One protein which we have termed PICK1 may be involved in PKC$\alpha$-specific function at the level of the nuclear membrane after activation. Binding of PICK1 to PKC$\alpha$ has been shown to be isoform specific as it does not bind to PKC$\beta$II or PKC$\alpha$ in the yeast two-hybrid system. PICK1 mRNA expression level is highest in testis and brain with lower levels of expression in skeletal muscle, heart, kidney, lung and liver. PICK1 protein contains five PKC consensus phosphorylation sites and serves as an in vitro substrate for PKC. The PICK1 protein also contains a P-Loop motif that has been shown to bind ATP or GTP in the Ras family of oncoproteins as well as the G-Protein family. Proteins which bind ATP or GTP using this motif all have some sort of catalytic function although none has been identified for PICK1 as yet. PICK1 contains a DHR/GLGF motif at the N-terminus of the protein. The DHR/GLGF motif is contained in a number of recently described proteins and has been shown to mediate protein-protein interactions at the level of membranes and cytoskeleton. When both PKC$\alpha$ and PICK1 are co-expressed in Cos1 cells the two proteins co-localize to the perinucleus in immunoflouresence studies and co-immunoprecipitate. The binding site for PKC7 has been localized to amino acids 1-358 on PICK1 which contains the DHR/GLGF motif. Binding of PICK1 to PKC$\alpha$ requires the hinge and C-terminal domains of PKC$\alpha$. In vitro, PICK1 binds to PKC$\alpha$ and inhibits its activity as assayed by myelin basic protein phosphorylation. PICK1 also binds to TIS21, a primary response gene that is expressed in response to phorbol ester and growth factor treatment. The Caenorhabditis elegans homologue of PICK1 has been cloned and sequenced revealing a high degree of conservation in the DHR/GLGF motif. A more C-terminal region also shows a high degree of conservation, and the C. elegans PICK1 homologue binds to PKC7 suggesting a conservation of function. Taken together these results suggest that PICK1 may be involved in a PKC$\alpha$-specific function at the level of the nuclear membrane. ^

drp, A novel cell density regulated protein

Growing cells are continuously processing signals of all varieties and responding to these signals by changes in cellular gene expression. One signal that cells in close proximity relay to each other is cell-cell contact. Non-transformed cells respond to cell-cell contact by arrest of growth and entry into G$\sb0,$ a process known as contact inhibition. Transformed cells do not respond to contact inhibition and continue to grow to high cell density, forming foci when in cell culture and tumors in the living organism. The events surrounding the generation, transduction, and response to cellular contact are poorly understood. In the present study, a novel gene product, drp, is shown to be expressed at high levels in cultured cells at high cell density. This density regulated protein, drp, has an apparent molecular weight of 70 kDa. Northern analysis shows drp to be highly expressed in cardiac and skeletal muscle and least abundant in lung and kidney tissues. By homology to two independently derived sequence tagged sites (STSs) used in the human genome project, drp or a closely related sequence maps to human chromosome 12. Density-dependent increases in drp expression have been demonstrated in six different cell lines including NIH 3T3, Hela and a human teratocarcinoma cell line, PA-1. Cells exhibit increased drp expression both when they are plated at increasing concentrations per unit area, or plated at low density and allowed to grow naturally to higher cell density. Cells at high density can exhibit several phenotypes including growth arrest, accumulation of soluble factors in the media, and increased numbers of cell contacts. Growth arrest by serum starvation or TGF-$\beta$ treatment fails to produce an increase in drp expression. Similarly, treatment of low density cells with conditioned media from high density cells fails to elicit drp expression. These results argue that neither soluble factors accumulated or expressed at high density nor simple exit from the cell cycle is sufficient to produce an increase in drp expression. The expression of drp appears to be uniquely regulated by cell density alone. ^

Analysis of pp60$\sp{{\rm c}{-}src}$ and pp62$\sp{{\rm c}{-}yes}$ intracellular protein tyrosine kinase function in colon tumor cell growth regulation using herbimycin A, a tyrosine kinase specific inhibitor

Two approaches were utilized to investigate the role of pp60c-src activation in growth control of model colon tumor cell lines. The first approach involved analysis of pp60c-src activity in response to growth factor treatment to determine if transient activation of the protein was associated with ligand induced mitogenic signal transduction as occurs in non-colonic cell types. Activation of pp60c-src was detected using colon tumor cell lysates after treatment with platelet derived growth factor (PDGF). Activation of pp60c-src was also detected in response to epidermal growth factor (EGF) treatment using cellular lysates and intact cells. In contrast, down-regulation of purified pp60c-src occurred after incubation with EGF-treated EGFr immune complexes in vitro suggesting additional cellular events were potentially required for the stimulatory response observed in intact cells. The results demonstrated activation of pp60c-src in colon tumor cells in response to PDGF and EGF which is consistent with the role of the protein in mitogenic signal transduction in non-colonic cell types.^ The second approach used to study the role of pp60c-src activation in colonic cell growth control focused on analysis of the role of constitutive activation of the protein, which occurs in approximately 80% of colon tumors and cell lines, in growth control. These studies involved analysis of the effects of the tyrosine kinase specific inhibitor Herbimycin A (HA) on monolayer growth and pp60c-src enzymatic activity using model colon tumor cell lines. HA induced dose-dependent growth inhibition of all colon tumor cell lines examined possessing elevated pp60c-src activity. In HT29 cells the dose-dependent growth inhibition induced by HA correlated with dose-dependent pp60c-src inactivation. Inactivation of pp60c-src was shown to be an early event in response to treatment with HA which preceded induction of HT29 colon tumor cell growth inhibition. The growth effects of HA towards the colon tumor cells examined did not appear to be associated with induction of differentiation or a cytotoxic mechanism of action as changes in morphology were not detected in treated cells and growth inhibition (and pp60c-src inactivation) were reversible upon release from treatment with the compound. The results suggested the constitutive activation of pp60c-src functioned as a proliferative signal in colon tumor cells. Correlation between pp60c-src inactivation and growth inhibition was also observed using HA chemical derivatives confirming the role of tyrosine kinase inactivation by these compounds in inhibition of mitogenic signalling. In contrast, in AS15 cells possessing specific antisense mRNA mediated inactivation of pp60c-src, HA-induced inactivation of the related pp62c-yes tyrosine kinase, which is also activated during colon tumor progression, was not associated with induction of monolayer growth inhibition. These results suggested a function for the constitutively activated pp62c-yes protein in colon tumor cell proliferation which was different from that of activated pp60c-src. (Abstract shortened by UMI.) ^

Immunology and HIV expression in skin, in vitro, and in response to ultraviolet light

HIV can enter the body through Langerhans cells, dendritic cells, and macrophages in skin mucosa, and spreads by lysis or by syncytia. Since UVL induces of HIV-LTR in transgenic mice mid in cell lines in vitro, we hypothesized that UVB may affect HIV in people and may affect HIV in T cells in relation to dose, apoptosis, and cytokine expression. To determine whether HIV is induced by UVL in humans, a clinical study of HIV+ patients with psoriasis or pruritus was conducted during six weeks of UVB phototherapy, Controls were HIV-psoriasis patients receiving UVB and HIV+ KS subjects without UVB.Blood and skin biopsy specimens were collected at baseline, weeks 2 and 6, and 4 weeks after UVL. AIDS-related skin diseases showed unique cytokine profiles in skin and serum at baseline. In patients and controls on phototherapy, we observed the following: (1) CD4+ and CD8+ T cell numbers are not significantly altered during phototherapy, (2) p24 antigen levels, and also HIV plasma levels increase in patients not on antiviral therapy, (3) HIV-RNA levels in serum or plasma. (viral load) can either increase or decrease depending on the patient's initial viral load, presence of antivirals, and skin type, (4) HIV-RNA levels in the periphery are inversely correlated to serum IL-10 and (5) HIV+ cell in skin increase after UVL at 2 weeks by RT-PCR in situ hybridization mid we negatively correlated with peripheral load. To understand the mechanisms of UVB mediated HIV transcription, we treated Jurkat T cell lines stably transfected with an HIV-LTR-luciferase plasmid only or additionally with tat-SV-40 early promoter with UVB (2 J/m2 to 200 J/m2), 50 to 200 ng/ml rhIL-10, and 10 μg/ml PHA as control. HIV promoter activity was measured by luciferase normalized to protein. Time points up to 72 hours were analyzed for HIV-LTR activation. HIV-LTR activation had the following properties: (1) requires the presence of Tat, (2) occurs at 24 hours, and (3) is UVB dose dependent. Changes in viability by MTS (3-(4,5-dimethyhhiazol-2-y1)-5-(3-carboxymethoxyphonyl)-2-(4-sulfophenyl)-2H-tetrazolium) mixed with PMS (phenazine methosulfate) solution and apoptosis by propidium iodide and annexin V using flow cytometry (FC) were seen in irradiated Jurkat cells. We determined that (1) rhIL-10 moderately decreased HIV-LTR activation if given before radiation and greatly decreases it when given after UVB, (2) HIV-LTR activation was low at doses of greater than 70 J/m2, compared to activation at 50 J/m2. (Abstract shortened by UMI.)^