Ca++/calmodulin-dependent protein kinase II: Messenger RNA in developing rat brain

Ca$\sp{++}$/calmodulin-dependent protein kinase II (CaM-KII) is highly concentrated in mammalian brain, comprising as much as 2% of the total protein in some regions. In forebrain, CaM-KII has been shown to be enriched in postsynaptic structures where it has been implicated in maintaining cytoskeletal structure, and more recently in signal transduction mechanisms and processes underlying learning and memory. CaM-KII appears to exist as a holoenzyme composed of two related yet distinct subunits, alpha and beta. The ratio of the subunits in the holoenzyme varies with different brain regions and to some degree with subcellular fractions. The two subunits also display distinct developmental profiles. Levels of alpha subunit are not evident at birth but increase dramatically during postnatal development, while levels of beta subunit are readily detected at birth and only gradual increase postnatally. The distinct regional, subcellular and developmental distribution of the two subunits of CaM-KII have prompted us to examine factors involved in regulating the synthesis of the subunit proteins.^ This dissertation addresses the regional and developmental expression of the mRNAs for the individual subunits using in situ hybridization histochemistry and northern slot-blot analysis. By comparing the developmental profile of each mRNA with that of its respective protein, we have determined that initiation of gene transcription is likely the primary site for regulating CaM-KII protein levels. Furthermore, the distinct cytoarchitecture of the hippocampus has allowed us to demonstrate that the alpha, but not beta subunit mRNA is localized in dendrites of certain forebrain neurons. The localization of alpha subunit mRNA at postsynaptic structures, in concert with the accumulation of subunit protein, suggests that dendritic synthesis of CaM-KII alpha subunit may be important for maintaining postsynaptic structure and/or function. ^

Structural determinants of subunit -subunit interactions and subcellular localization of the calcium /calmodulin -dependent protein kinase II

The multifunctional Ca$\sp{2+}$/calmodulin-dependent protein kinase II (CaM kinase) is a Ser/Thr directed protein kinase that participates in diverse Ca$\sp{2+}$ signaling pathways in neurons. The function of CaM kinase depends upon the ability of subunits to form oligomers and to interact with other proteins. Oligomerization is required for autophosphorylation which produces significant functional changes that include Ca$\sp{2+}$/calmodulin-independent activity and calmodulin trapping. Associations with other proteins localize CaM kinase to specific substrates and effectors which serves to optimize the efficiency and speed of signal transduction. In this thesis, we investigate the interactions that underlie the appropriate positioning of CaM kinase activity in cells. We demonstrate that the subcellular distribution of CaM kinase is dynamic in hippocampal slices exposed to anoxic/aglycemic insults and to high K$\sp{+}$-induced depolarization. We determine the localization of CaM kinase domains expressed in neurons and PC-12 cells and find that the C-terminal domain of the $\alpha$ subunit is necessary for localization to dendrites. Moreover, monomeric forms of the enzyme gain access to the nucleus. Attempts made to identify novel CaM kinase binding proteins using the yeast two-hybrid system resulted in the isolation of hundreds of positive clones. Those that have been sequenced are identical to CaM kinase isoforms. Finally, we report the discovery of specific regions within the C-terminal domain that are necessary and sufficient for subunit-subunit interactions. Differences between the $\alpha$ and $\beta$ isoforms were discovered that indicate unique structural requirements for oligomerization. A model for how CaM kinase subunits interact to form holoenzymes and how structural heterogeneity might influence CaM kinase function is presented. ^

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II.

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.

Multisite phosphorylation of ornithine decarboxylase in transformed macrophages

Ornithine decarboxylase (ODC), the initial inducible enzyme in the polyamine biosynthetic pathway, exists in the transformed macrophage RAW264 cell line as a phosphoprotein following cell stimulation. The hypothesis that ODC is phosphorylated at multiple sites in stimulated RAW264 cells was investigated. ODC isolated from tetradecanoyl-phorbol-13-acetate (TPA)-stimulated cells metabolically radiolabeled in the presence of $\sp{32}$P$\sb{\rm i}$ was subjected to cyanogen bromide (CNBr) cleavage followed by phosphopeptide mapping and two dimensional phosphoamino acid analysis. These phosphorylation studies demonstrated six in situ phosphorylated CNBr-generated fragments having apparent molecular weights of 17, 14.3, 8, 6.5, 4, and 2.7 kDa and also revealed that ODC is phosphorylated in RAW264 cells on at least 5 serine and 2 threonine residues.^ In addition, the in vivo specific activity and phosphorylation pattern of ODC in response to various kinase cascade stimulants was studied. A differential response in ODC specific activity and a variation in the relative distribution of $\sp{32}$P-labeling of serine and threonine residues on the ODC molecule was noted in response to fetal bovine serum, cAMP and isobutylmethylxanthine, lipopolysaccharide, or TPA.^ Based on information derived from consensus sequence motifs, three protein kinases responsible for the phosphorylation of ODC in vitro were identified. Purified ODC was phosphorylated in vitro by casein kinase II (CK II), extracellular signal-regulated kinase 1 (ERK1), and its activator, extracellular signal-regulated kinase kinase (MEK). CK II phosphorylated ODC on serine residues contained on three CNBr-generated peptides with apparent molecular weights of 14.3, 6.5, and 2.7 kDa. Both ERK1 and MEK phosphorylated ODC on serine and threonine residues on a CNBr-generated peptide fragment with an apparent molecular weight of 6.5 kDa. The in vitro radiolabeled peptides corresponded in molecular mass with some of the CNBr fragments of ODC phosphorylated in situ in stimulated RAW264 cells.^ This study concludes that ODC is phosphorylated in the transformed macrophage RAW264 cell line at multiple sites in response to various kinase cascade stimulants. These stimulants also led to a differential response in specific activity and phosphorylation pattern of ODC in RAW264 cells. Three protein kinases have been identified which phosphorylate ODC in vitro on peptides and amino acid residues which correspond with those phosphorylated in situ. ^

Inactivation and self -association of CaM kinase: Effects of ATP, substrate binding domains, and isozymic forms

Calcium/calmodulin-dependent protein kinase II (CaM kinase) is a multifunctional Ser/Thr protein kinase, that is highly enriched in brain and is involved in regulating many aspects of neuronal function. We observed that forebrain CaM kinase from crude homogenates, cytosolic fractions and purified preparations inactivates and translocates into the particulate fraction following autophosphorylation. Using purified forebrain CaM kinase as well as recombinant $\alpha$ isozyme, we determined that the formation of particulate enzyme was due to enzyme self-association. The conditions of autophosphorylation determine whether enzyme self-association and/or inactivation will occur. Self-association of CaM kinase is sensitive to pH, ATP concentration, and enzyme autophosphorylation. This process is prevented by saturating concentrations of ATP. However, in limiting ATP, pH is the dominant factor, and enzyme self-association occurs at pH values $\rm{<}7.0.$ Site-specific mutants were produced by substituting Ala for Thr286, Thr253, or Thr305,306 to determine whether these sites of autophosphorylation affect enzyme inactivation and self-association. The only mutation that influenced these processes was Ala286, which removed the protective effect afforded by autophosphorylation in saturating ATP. Enzyme inactivation occurs in the presence and absence of self-association and appears predominantly sensitive to nucleotide concentration, because saturating concentrations of $\rm Mg\sp{2+}/ADP$ or $\rm Mg\sp{2+}/ATP$ prevent this process. These data implicate the ATP binding pocket in both inactivation and self-association. We also observed that select peptide substrates and peptide inhibitors modeled after the autoregulatory domain of CaM kinase prevented these processes. The $\alpha$ and $\beta$ isozymes of CaM kinase were characterized independently, and were observed to exhibit differences in both enzyme inactivation and self-association. The $\beta$ isozyme was less sensitive to inactivation, and was never observed to self-associate. Biophysical characterization, and transmission electron microscopy coupled with image analysis indicated both isozymes were multimeric, however, the $\alpha$ and $\beta$ isozymes appeared structurally different. We hypothesize that the $\alpha$ subunit of CaM kinase plays both a structural and enzymatic role, and the $\beta$ subunit plays an enzymatic role. The ramifications for the functional differences observed for inactivation and self-association are discussed based on potential structural differences and autoregulation of the $\alpha$ and $\beta$ isozymes in both calcium-induced physiological and pathological processes. ^

Long-term synaptic facilitation of tail sensorimotor connections in {\it Aplysia\/}

Long-term sensitization in Aplysia is a well studied model for the examination of the cellular and molecules mechanisms of long-term memory. Several lines of evidence suggest long-term sensitization is mediated at least partially by long-term synaptic facilitation between the sensory and motor neurons. The sensitization training and one of its analogues, serotonin (5-HT), can induce long-term facilitation. In this study, another analogue to long-term sensitization training has been developed. Stimulation of peripheral nerves of pleural-pedal ganglia preparation induced long-term facilitation at both 24 hr and 48 hr. This is the first report that long-term facilitation in Aplysia persists for more than 24 hr, which is consistent with the observation that long-term sensitization lasts for more than one day. Thus, the data support the hypothesis that long-term facilitation is an important mechanism for long-term sensitization.^ One of the major differences between short-term and long-term facilitation is that long-term facilitation requires protein synthesis. Therefore, the effects of anisomycin, a protein synthesis inhibitor, on long-term facilitation was examined. Long-term facilitation induced by nerve stimulation was inhibited by 2 $\mu$M anisomycin, which inhibits $\sim$90% of protein synthesis. Nevertheless, at higher concentration (20 $\mu$M), anisomycin induced long-term facilitation by itself, which raises an interesting question about the function of anisomycin other than protein synthesis inhibition.^ Since protein synthesis is critical for long-term facilitation, a major goal is to identify and functionally characterize the molecules whose mRNA levels are altered during the formation of long-term facilitation. Behavioral training or its analogues (nerve stimulation and 5-HT) increases the level of mRNA of calmodulin (CaM). Thus, the role of Ca$\sp{2+}$-CaM-dependent protein kinase II (CaMKII), a major substrate of CaM, in long-term facilitation induced by nerve stimulation was examined. KN-62, a specific CaMKII inhibitor, did not block either the induction or the maintenance of long-term facilitation induced by nerve stimulation. These data indicate that CaMKII may not be involved in long-term facilitation. Another protein whose mRNA level of a molecule was increased by the behavioral training and the treatment of 5-HT is Aplysia tolloid/BMP-1-like protein 1 (apTBL-1). Tolloid in Drosophila and BMP-1 in human tissues are believed to be secreted as a metalloprotease to activate TGF-$\beta.$ Thus, the long-term effects of recombinant human TGF-$\beta1$ on synaptic strength were examined. Treatment of ganglia with TGF-$\beta1$ produced long-term facilitation, but not short-term or intermediate-term facilitation ($\le$4 hr). In addition, TGF-$\beta1$ and 5-HT were not additive in producing long-term facilitation, which indicates an interaction between two cascades. Moreover, 5-HT-induced facilitation (at both 24 hr and 48 hr) and nerve stimulation-induced facilitation (at 24 hr) were inhibited by TGF-$\beta$ sRII, a TGF-$\beta$ inhibitor. These results suggest that TGF-$\beta$ is part of the cascade of events underlying long-term sensitization, and also indicate that a signaling molecule used in development may also have functions in adult neuronal plasticity. ^

SIGNALING MECHANISMS THAT CONTROL GAP JUNCTIONAL COUPLING BETWEEN RETINAL NEURONS

Gap junctions between neurons form the structural substrate for electrical synapses. Connexin 36 (Cx36, and its non-mammalian ortholog connexin 35) is the major neuronal gap junction protein in the central nervous system (CNS), and contributes to several important neuronal functions including neuronal synchronization, signal averaging, network oscillations, and motor learning. Connexin 36 is strongly expressed in the retina, where it is an obligatory component of the high-sensitivity rod photoreceptor pathway. A fundamental requirement of the retina is to adapt to broadly varying inputs in order to maintain a dynamic range of signaling output. Modulation of the strength of electrical coupling between networks of retinal neurons, including the Cx36-coupled AII amacrine cell in the primary rod circuit, is a hallmark of retinal luminance adaptation. However, very little is known about the mechanisms regulating dynamic modulation of Cx36-mediated coupling. The primary goal of this work was to understand how cellular signaling mechanisms regulate coupling through Cx36 gap junctions. We began by developing and characterizing phospho-specific antibodies against key regulatory phosphorylation sites on Cx36. Using these tools we showed that phosphorylation of Cx35 in fish models varies with light adaptation state, and is modulated by acute changes in background illumination. We next turned our focus to the well-studied and readily identifiable AII amacrine cell in mammalian retina. Using this model we showed that increased phosphorylation of Cx36 is directly related to increased coupling through these gap junctions, and that the dopamine-stimulated uncoupling of the AII network is mediated by dephosphorylation of Cx36 via protein kinase A-stimulated protein phosphatase 2A activity. We then showed that increased phosphorylation of Cx36 on the AII amacrine network is driven by depolarization of presynaptic ON-type bipolar cells as well as background light increments. This increase in phosphorylation is mediated by activation of extrasynaptic NMDA receptors associated with Cx36 gap junctions on AII amacrine cells and by Ca2+-calmodulin-dependent protein kinase II activation. Finally, these studies indicated that coupling is regulated locally at individual gap junction plaques. This work provides a framework for future study of regulation of Cx36-mediated coupling, in which increased phosphorylation of Cx36 indicates increased neuronal coupling.

A model of the roles of essential kinases in the induction and expression of late long-term potentiation.

The induction of late long-term potentiation (L-LTP) involves complex interactions among second-messenger cascades. To gain insights into these interactions, a mathematical model was developed for L-LTP induction in the CA1 region of the hippocampus. The differential equation-based model represents actions of protein kinase A (PKA), MAP kinase (MAPK), and CaM kinase II (CAMKII) in the vicinity of the synapse, and activation of transcription by CaM kinase IV (CAMKIV) and MAPK. L-LTP is represented by increases in a synaptic weight. Simulations suggest that steep, supralinear stimulus-response relationships between stimuli (e.g., elevations in [Ca(2+)]) and kinase activation are essential for translating brief stimuli into long-lasting gene activation and synaptic weight increases. Convergence of multiple kinase activities to induce L-LTP helps to generate a threshold whereby the amount of L-LTP varies steeply with the number of brief (tetanic) electrical stimuli. The model simulates tetanic, -burst, pairing-induced, and chemical L-LTP, as well as L-LTP due to synaptic tagging. The model also simulates inhibition of L-LTP by inhibition of MAPK, CAMKII, PKA, or CAMKIV. The model predicts results of experiments to delineate mechanisms underlying L-LTP induction and expression. For example, the cAMP antagonist RpcAMPs, which inhibits L-LTP induction, is predicted to inhibit ERK activation. The model also appears useful to clarify similarities and differences between hippocampal L-LTP and long-term synaptic strengthening in other systems.

The role of post-translation regulation of Twist expression in the tumor progression of squamous cell carcinoma of head and neck

Squamous cell carcinoma of head and neck (SCCHN) is the tenth most common cancer in the world. Unfortunately, the survival of patients with SCCHN has not improved in the last 40 years. Therefore new targets for therapy are needed, and to this end we are studying signaling pathways activated by IL-6 which we have found stimulates cell migration and soft agar growth in SCCHN. Our data show that IL-6 increases TWIST expression in a transcription-independent mechanism in many SCCHN cell lines. Further investigation reveals TWIST can be phosphorylated upon IL-6 treatment. By computation prediction (http://scansite.mit.edu/motifscan_seq.phtml ), we found that TWIST has a putative phosphorylation site for casein kinase 2 (CK2) suggesting that this kinase could serve as a link between IL-6 stimulation and Twist stability. To test this hypothesis, we used a CK2 inhibitor and shRNA to CK2 and found that these interventions inhibited IL-6 stimulation of TWIST stability. In addition, mutation of the putative CK2 phosphorylation site (S18/S20A) in TWIST decreased the amount of phospho-ATP incorporated by TWIST in an in vitro kinase assay, and altered TWIST stability. In Boyd chamber migration assay and wound-healing assay, the CK2 inhibitor, DMAT, was found to decrease the motility of IL-6 stimulated SCCHN cells and over expression of either a wild-type or the hyperphosphorylated mimicking mutant S18/20D –Twist rather than the hypo-phosphorylated mimicking mutant S18/20A-Twist can promote SCCHN cell motility.To our knowledge, this is the first report to identify the importance of IL-6 stimulated CK2 phosphorylation of TWIST in SCCHN. As CK2 inhibitors are currently under phase I clinical trials, our findings indicate that CK2 may be a viable therapeutic target in SCCHN. Therefore, further pre-clinical studies of this inhibitor are underway.

Evolutionarily conserved role of calcineurin in phosphodegron-dependent degradation of phosphodiesterase 4D.

Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin. Here we uncover a novel regulatory pathway for cyclic AMP (cAMP) signaling by the phosphatase calcineurin which is also evolutionarily conserved in Caenorhabditis elegans. We found that calcineurin binds directly to and inhibits the proteosomal degradation of cAMP-hydrolyzing phosphodiesterase 4D (PDE4D). We show that ubiquitin conjugation and proteosomal degradation of PDE4D are controlled by a cullin 1-containing E(3) ubiquitin ligase complex upon dual phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3beta (GSK3beta) in a phosphodegron motif. Our findings identify a novel signaling process governing G-protein-coupled cAMP signal transduction-opposing actions of the phosphatase calcineurin and the CK1/GSK3beta protein kinases on the phosphodegron-dependent degradation of PDE4D. This novel signaling system also provides unique functional insights into the complications elicited by CsA in transplant patients.

Lobe specific Ca2+-calmodulin nano-domain in neuronal spines: a single molecule level analysis.

Calmodulin (CaM) is a ubiquitous Ca(2+) buffer and second messenger that affects cellular function as diverse as cardiac excitability, synaptic plasticity, and gene transcription. In CA1 pyramidal neurons, CaM regulates two opposing Ca(2+)-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD). Induction of LTP and LTD require activation of Ca(2+)-CaM-dependent enzymes: Ca(2+)/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca(2+) and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca(2+) ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca(2+) and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca(2+) signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca(2+)-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca(2+) channels, and to the microscopic injection rate of Ca(2+) ions. We also demonstrate that Ca(2+) saturation takes place via two different pathways depending on the Ca(2+) injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca(2+) sensors that can differentially transduce Ca(2+) influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca(2+)-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity.

A study in the molecular and cellular functions of GSK3beta in prostate adenocarcinoma

Kinases are part of a complex network of signaling pathways that enable a cell to respond to changes in environmental conditions in a regulated and coordinated way. For example, Glycogen Synthase Kinase 3 beta (GSK3β) modulates conformational changes, protein-protein interaction, protein degradation, and activation of unique domains in proteins that transduce signals from the extracellular milieu to the nucleus. ^ In this project, I investigated the expression and function that GSK3β exhibits in prostate cells. The capacity of GSK3β to regulate two transcription factors (JUN and CREB), which are known to be inversely utilized in prostate tumor cells, was measured. JUN/AP1 is constitutively activated in PC-3 cells; whereas, CREB/CRE activity is ∼20 fold less than the former. GSK3β overexpression obliterates JUN/AP1 activity. With respect to CREB GSK3β increases CREB/CRE activity. Cellular levels of active GSK3β can determine whether JUN or CREB is preferentially active in the PC-3s. Theoretically, in response to a particular cellular context or stimulus, a cell may coordinate JUN and CREB function by regulating GSK3β.^ A comparison of various prostate cell lines showed that active GSK3β is less expressed in normal prostate epithelial cells than in tumor cells. Differentially expressed active (GSK3β) may correlate with progression of prostate carcinoma. If a known marker associated with carcinoma of the prostate could be shown to be regulated by GSK3β then, further study of GSK3β may lead to a better understanding of both possible prevention of the disease and improved therapy for advanced stages. ^ The androgen receptor (AR) is an intriguing phosphoprotein whose regulation is potentially determined by a variety of kinases. One of these is (GSK3β) I found that (GSK3β) is a regulator of the androgen receptor in both the unliganded and liganded states. It can inhibit AR function as measured by reporter assays. Also, GSK3β associates with the AR at the DNA binding domain because deletion constructs expressing either the n-terminus or the c-terminus (both having the DBD in common) immunoprecipitated with GSK3β. Increased understanding of how GSK3β functions in prostate cancer would provide clues into how (1) certain signal pathways are coordinated and (2) the androgen receptor may be regulated. ^

A novel mechanism for regulation of calmodulin signaling by RC3

RC3, also known as neurogranin, is a small neuronal IQ domain protein whose only known function is to bind calmodulin (CaM). The hypothesis tested in this work was that RC3 alters the dynamics of the interaction of Ca 2+-CaM with CaM-kinase II, so that there is less CaM-kinase II activation for a given Ca2+ stimulus. To evaluate this hypothesis, we investigated the affinity and kinetics of the interactions of CaM with Ca 2+, RC3 and CaM-kinase II. We quantitated the interaction of the four CaM-kinase II isoforms with CaM and found that the KD for binding of CaM to CaM-kinase II ranged from 7 nM to 60 nM. Using stopped-flow fluorimetry, we determined the kinetics of the interaction of Ca2+-CaM with αCaM-kinase II, and found that the association rate constant is 2.1 × 10 M −1s−1 and the dissociation rate constant is 1.6 s−1. We investigated the effects of RC3 and αCaM-kinase II on the affinity of CaM for Ca2+ and found that both proteins alter the rate of dissociation of Ca2+ from CaM. RC3 increases the rate of dissociation of Ca2+ from the C-terminal binding sites of CaM from 9 s−1 to ∼500 s−1 , while αCaM-kinase II causes a decrease in the rate of dissociation from all four Ca2+ binding sites. Measurement of the rate of dissociation of Ca2+ from CaM in the presence of both RC3 and αCaM-kinase II revealed a role for RC3 in accelerating the dissociation of the Ca 2+-CaM-αCaM-kinase II complex at the end of a Ca2+ signal. We characterized the interaction of RC3 with apo-CaM and Ca 2+-CaM and found that the KD for both of these interactions is about 1 μM. We also directly tested whether RC3 slowed the dynamics of the binding of CaM to αCaM-kinase II and found that RC3 had no effect for large changes in Ca2+, and a modest effect for small changes in Ca2+ levels. Our overall conclusion is that the ability of RC3 to alter the interaction of Ca2+ with CaM allows RC3 to alter the dynamics of interaction of CaM with Ca2+-dependent targets such as CaM-kinase II. ^

LY2109761, a novel transforming growth factor beta receptor type I and type II dual inhibitor, as a therapeutic approach to suppressing pancreatic cancer metastasis.

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor beta (TGF-beta) plays a key role in cancer metastasis, signaling through the TGF-beta type I/II receptors (TbetaRI/II). We hypothesized that targeting TbetaRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-beta1-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TbetaRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TbetaRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis.

Regulation of adenylyl cyclase by protein kinase C and P$\sb2$ purinergic agonists

Activation of protein kinase C (PKC) causes multiple effects on adenylyl cyclase (AC), (i) an inhibition of (hormone) receptor/G$\sb{\rm s}$ coupling, consistent with PKC modification of the receptor and (ii) a postreceptor sensitization consistent with a PKC-mediated modification of the stimulatory (G$\sb{\rm s}$) or inhibitory (G$\sb{\rm i}$) G-proteins or the catalyst (C) of AC. In L cells expressing the wild-type beta-adrenergic receptor ($\beta$AR) 4-$\beta$ phorbol 12-myristate-13-acetate (PMA) caused 2-3-fold increases in the K$\sb{\rm act}$ and V$\sb{\rm max}$ for epinephrine-stimulated AC activity and an attenuation of GTP-mediated inhibition of AC. Deletion of a concensus site for PKC phosphorylation (amino acids 259-262) from the $\beta$AR eliminated the PMA-induced increase in the K$\sb{\rm act}$, but had no effect on the other actions of PMA. PMA also increased the K$\sb{\rm act}$ and V$\sb{\rm max}$ for prostaglandin E$\sb1$ (PGE$\sb1$)-stimulated AC and the V$\sb{\rm max}$ for forskolin-stimulated AC. Maximal PMA-induced sensitizations were observed when AC was assayed in the presence of 10 $\mu$M GTP and 0.3 mM (Mg$\sp{++}$).^ Liao et al. (J. Biol. Chem. 265:11273-11284 (1990)) have shown that the P$\sb2$ purinergic receptor agonist ATP stimulates hydrolysis of 4,5 inositol bisphosphate (PIP$\sb2$) by phospholipase C (PLC) in L cells. To determine if agonists that stimulate PLC and PMA had similar effects on AC function we compared the effects of ATP and PMA. ATP caused a rapid 50-150% sensitization of PGE$\sb1$-, epinephrine-, and forskolin-stimulated AC activity with an EC$\sb{50}$ of 3 $\mu$M ATP. The sensitization was similar (i.e. Mg$\sp{++}$ and GTP sensitivity) to that caused by 10 nM PMA. However, unlike PMA ATP did not affect the K$\sb{\rm act}$ for hormone-stimulated AC and its effects were unaltered by down-regulation of PKCs following long term PMA treatment. Our results demonstrate that a PKC concensus site in the $\beta$AR, is required for the PMA-induced decrease in receptor/G$\sb{\rm s}$ coupling. Our data also indicate that activation of P$\sb2$ purinergic receptors by ATP may be important in the sensitization of AC in L cells. The mechanism behind this effect remains to be determined. ^