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. ^

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.

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.

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. ^

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. ^

Role of protein kinase C in short-term sensitization of {\it Aplysia}

Plasticity at the connections between sensory neurons and their follower cells in Aplysia has been used extensively as a model system to examine mechanisms of simple forms of learning, such as sensitization. Sensitization is induced, at least in part, by the transmitter serotonin (5-HT) and expressed in several forms, including facilitation of sensorimotor connections. Spike broadening has been believed to be a key mechanism underlying facilitation of nondepressed synapses. Previously, this broadening was believed to be dependent primarily on cAMP/protein kinase A (PKA)-mediated reduction of a noninactivating, relatively voltage-independent K$\sp{+}$ current termed the S-K$\sp+$ current (I$\sb{\rm K{,}S}$). Recent evidence, however, suggests that 5-HT-induced somatic spike broadening is composed of at least two components: a cAMP-dependent, rapidly developing component and a cAMP-independent, slowly developing component.^ Phorbol esters, activators of protein kinase C (PKC), mimicked the cAMP-independent component of 5-HT-induced broadening. Staurosporine, which inhibits PKC, had little effect on the rapidly developing component of 5-HT-induced broadening, but inhibited significantly the slowly developing component. These results suggest that PKC is involved in the cAMP-independent component of 5-HT-induced broadening. The membrane currents responsible for the slowly developing component of broadening were examined. Activation of PKC mimicked, and partially occluded, 5-HT-induced modulation of membrane currents above 0 mV, where a voltage-dependent K$\sp+$ current (I$\sb{\rm K{,}V}$) is significantly activated. This modulation was complex because it was associated with a reduction in the magnitude of I$\sb{\rm K{,}V}$, as well as a slowing of both activation and inactivation kinetics of I$\sb{\rm K{,}V}$. These results support the hypothesis that PKC modulates I$\sb{\rm K{,}V}$ and that this modulation contributes to the slowly developing component of 5-HT-induced broadening. Based on these results and others, a new scheme for 5-HT-induced spike broadening is proposed in which the modulatory effects are mediated via two second messenger/protein kinase systems converging and diverging on multiple ionic conductances.^ The relationship between spike broadening and synaptic facilitation was also examined. Pharmacological reduction of I$\sb{\rm K{,}V}$ by low concentrations of 4-aminopyridine (4-AP) led to spike broadening and facilitation of the nondepressed sensorimotor connections, indicating that spike broadening via the reduction of I$\sc{K,V}$ can facilitate the synaptic connection. Further analyses, however, revealed that 4-AP-induced facilitation has qualitative differences from 5-HT- and PKC-induced facilitation. These results suggest that 5-HT- and PKC-induced facilitation of nondepressed synapses is mediated, at least in part, by spike-duration independent (SDI) processes. Under certain conditions, the PKC inhibitor, staurosporine, significantly inhibited the 5-HT-induced facilitation of sensorimotor connections.^ Finally, it was found that activation of PKC increased a basal level of cAMP and that PKC caused desensitization of the 5-HT receptor, which may be a possible negative feedback mechanism through which an extracellular ligand, 5-HT, is regulated. These results suggest that these two second messenger/protein kinase pathways can interact in the sensory neuron. Thus, neuronal plasticity that may contribute to learning and memory appears to involve several complex and interactive processes. ^

Rescue from apoptosis in early (CD34 selected) versus late (CD34 unselected) human hematopoietic cells by VLA-4 and VCAM-1 dependent adhesion to bone marrow stromal cells

The interaction of hematopoietic precursor cell with bone marrow stromal cells is assumed to be important to the survival of hematopoietic precursor cells during hematopoietic cell long-term culture. Early hematopoietic stem cells are preferentially found within the stromal adherent cell fraction in primary long-term bone marrow cultures. The purpose of this dissertation was to understand the molecular mechanisms that govern these interactions for the regulation of survival and proliferation of early versus late hematopoietic cells.^ Monoclonal antibodies to the VLA-4 recognize the alpha4 beta1 integrin receptor on human hematopoietic cells. This monoclonal antibody blocks the adhesion between early hematopoietic progenitor cells (CD34 selected cells) and stromal cells when added to cultures of these cells. Addition of the VLA-4 monoclonal antibody to cultures of stromal cells and CD34 selected cells was shown to induce apoptosis of CD34 selected cells in these CD34 selected cell/stromal cell cocultures, as measured by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling method. In contrast to these experiments with early hematopoietic progenitor cells (CD34+), the level of adhesion between more differentiated cells (unfractionated hematopoietic cells) and stromal cells was not significantly altered by addition of the anti-VLA-4 monoclonal antibody. Similarly, the level of apoptosis of unfractionated hematopoietic cells was not significantly increased by the addition of anti-VLA-4 monoclonal antibody to cultures of the latter cells with stromal cells. The binding of the unfractionated cells is less than that of the CD34 selected. Since there is no difference between the alpha4 beta1 integrin expression level of the early and late myeloid cells, there may be a difference in the functional state of the integrin between the early and late myeloid cells. We also show that CD34+ selected precursor cells proliferate at a higher rate when these cells are plated on recombinant VCAM-1 molecules. These data indicate that the alpha4beta1 integrin receptor (VLA-4) plays a central role in the apoptosis rescue function which results from the anchorage-dependent growth of the CD34 selected early hematopoietic cells on stromal cells. The data suggest that these apoptosis rescue pathways have less significance as the cells mature and become anchorage-independent in their growth. These data should assist in the design of systems for the ex vivo proliferation and transduction of early hematopoietic cells for genetic therapy. ^

p21 activated kinase 5 links multiple GTPase signaling pathways at mitochondria

The p21-activated kinase 5 (PAK5) is a serine/threonine protein kinase associated with the group 2 subfamily of PAKs. Although our understanding about PAK5 is very limited, it is receiving increasing interest due to its tissue specific expression pattern and important signaling properties. PAK5 is highly expressed in brain. Its overexpression induces neurite outgrowth in neuroblastoma cells and promotes survival in fibroblasts. ^ The serine/threonine protein kinase Raf-1 is an essential mediator of Ras-dependent signaling that controls the ERK/MAPK pathway. In contrast to PAK5, Raf-1 has been the subject of intensive investigation. However due to the complexity of its activation mechanism, the biological inputs controlling Raf-1 activation are not fully understood. ^ PAKs 1-3 are the known kinases responsible for phosphorylation of Raf-1 on serine 338, which is a crucial phosphorylation site for Raf-1 activation. However, dominant negative versions of these kinases do not block EGF-induced Raf-1 activation, indicating that other kinases may regulate the phosphorylation of Raf-1 on serine 338. ^ This thesis work was initiated to test whether the group 2 PAKs 4, 5 and 6 are responsible for EGF-induced Raf-1 activation. We found that PAK5, and to a lesser extent PAK4, can activate Raf-1 in cells. Our studies thereafter focused on PAK5. With the progress of our study we found that PAK5 does not significantly stimulate serine 338 phosphorylation of Triton X-100 soluble Raf-1. PAK5, however, constitutively and specifically associates with Raf-1 and targets it to a Triton X-100 insoluble, mitochondrial compartment, where PAK5 phosphorylates serine 338 of Raf-1. We further demonstrated that endogenous PAK5 and Raf-1 colocalize in Hela cells at the mitochondrial outer membrane. In addition, we found that the mitochondria-targeting of PAK5 is determined by its C-terminal kinase domain plus the upstream proximal region, and facilitated by the N-terminal p21 binding domain. We also demonstrated that Rho GTPases Cdc42 and RhoD associate with and regulate the subcellular localization of PAK5. Taken together, this work suggests that the mitochondria-targeting of PAK5 may link Ras and Rho GTPase-mediated signaling pathways, and sheds light on aspects of PAK5 signaling that may be important for regulating neuronal homeostasis. ^

Caenorhabditis elegans germinal center kinase (Gck-1) is a p-body component that inhibits precocious ectopic MAP kinase dependent meiotic maturation

The Caenorhabditis elegans germline is an excellent model system for studying meiosis, as the gonad contains germ cells in all stages of meiosis I prophase in a linear temporal and spatial pattern. To form healthy gametes, many events must be coordinated. Failure of any step in the process can reduce fertility. Here, we describe a C. elegans Germinal Center Kinase, GCK-1, that is essential for the accurate progression of germ cells through meiosis I prophase. In the absence of GCK-1, germ cells undergo precocious maturation due to the activation of a specific MAP kinase isoform. Furthermore, GCK-1 localizes to P-bodies, RNP particles that have been implicated in RNA degradation and translational control. Like two other components of C. elegans germline P-bodies, GCK-1 functions to limit physiological germ cell apoptosis. This is the first study to identify a role for a GCK-III kinase in metazoan germ cell development and to link P-body function with MAP kinase activation and germ cell maturation. ^

Reactive oxygen species-dependent mechanisms of N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in human carcinoma cells

4HPR is a synthetic retinoid that has shown chemopreventive and therapeutic efficacy against premalignant and malignant lesions including oral leukoplakia, ovarian and breast cancer, and neuroblastoma. 4HPR induces apoptosis in various cancer cells and production of reactive oxygen species (ROS) has been suggested as a possible cause underlying these effects. However, the mechanisms governing these effects by 4HPR are not fully elucidated. In this study, we explored the mechanisms of 4HPR-induced ROS increase and apoptosis in human cancer cells. ^ First, we identified genes modulated by 4HPR using oligonucleotide gene expression arrays and found that they fall into specific functional canonical pathways and gene networks using Ingenuity Pathways Analysis®. Further analysis has shown that 4HPR induced up-regulation of Endoplasmic Reticulum (ER)-related genes such as Heat shock proteins 70 and 90 and the transcriptional factor, GADD153. These findings were validated using quantitative real-time PCR. ^ Second, we found that 4HPR induced extensive ER stress evidenced by dilation of the ER and endoribonuclease-mediated splicing and activation of the transcriptional factor, XBP-1. In addition, 4HPR induced the up-regulation of various ER stress-related genes and their protein products, as well as cleavage and activation of the ER specific Caspase-4. Concomitantly with XBP-1 splicing, all of these effects were dependent on ROS generation by 4HPR. Furthermore, chemical inhibition and RNA interference studies revealed a novel pro-apoptotic role for HSP70/A1A in 4HPR-mediated apoptosis. ^ Third, we observed rapid activation of the small GTPase Rac by 4HPR which was upstream of ROS generation. Inhibition of Rac activity or silencing of its expression by RNA interference inhibited ROS generation and apoptosis induction by 4HPR. siRNA targeting PAK1 and expression of a dominant negative Rac, decreased 4HPR-mediated ROS generation, while expression of a constitutive active Rac increased basal and 4HPR-induced ROS generation and PARP cleavage. Furthermore, metastatic cancer cells exhibited higher Rac activation, ROS generation, and cell growth inhibition due to 4HPR exposure compared to their primary cancer cell counterparts. ^ These findings provide novel insights into 4HPR-mediated ROS generation and apoptosis induction and support the use of ROS inducing agents such as 4HPR against metastatic cancer cells. ^

Identification and analysis of novel mitotic inhibitors of the Caenorhabditis elegans Aurora B kinase

Proper execution of mitosis requires the accurate segregation of replicated DNA into each daughter cell. The highly conserved mitotic kinase AIR-2/Aurora B is a dynamic protein that interacts with subsets of cofactors and substrates to coordinate chromosome segregation and cytokinesis in Caenorhabdiris elegans. To identify components of the AIR-2 regulatory pathway, a genome-wide RNAi-based screen for suppressors of air-2 temperature-sensitive mutant lethality was conducted. Here, I present evidence that two classes of suppressors identified in this screen are bona fide regulators of the AIR-2 kinase. The strongest suppressor cdc-48.3, encodes an Afg2/Spaf-related Cdc48-like AAA+ ATPase that regulates AIR-2 kinase activity and stability during C. elegans embryogenesis. Loss of CDC-48.3 suppresses the lethality of air-2 mutant embryos, marked by the restoration of the dynamic behavior of AIR-2 and rescue of chromosome segregation and cytokinesis defects. Loss of CDC-48.3 leads to mitotic delays and abnormal accumulation of AIR-2 during late telophase/mitotic exit. In addition, AIR-2 kinase activity is significantly upregulated from metaphase through mitotic exit in CDC-48.3 depleted embryos. Inhibition of the AIR-2 kinase is dependent on (1) a direct physical interaction between CDC-48.3 and AIR-2, and (2) CDC-48.3 ATPase activity. Importantly, the increase in AIR-2 kinase activity does not correlate with the stabilization of AIR-2 in late mitosis. Hence, CDC-48.3 is a bi-functional inhibitor of AIR-2 that is likely to act via distinct mechanisms. The second class of suppressors consists of psy-2/smk-1 and pph-4.1, which encode two components of the conserved PP4 phosphatase complex that is essential for spindle assembly, chromosome segregation, and overall mitotic progression. AIR-2 and its substrates are likely to be targets of this complex since mitotic AIR-2 kinase activity is significantly increased during mitosis when either PSY-2/SMK-1 or PPH-4.l is depleted. Altogether, this study demonstrates that during the C. elegans embryonic cell cycle, regulators including the CDC-48.3 ATPase and PP4 phosphatase complex interact with and control the kinase activity, targeting behavior and protein stability of the Aurora B kinase to ensure accurate and timely progression of mitosis. ^

Role of activation of the protein tyrosine kinase, Src, in colorectal cancer chemoresistance

Advances in therapy for colorectal cancer have been hampered by development of resistance to chemotherapy. The Src family of protein tyrosine kinases has been associated with colorectal cancer development and progression. Activation of the prototypic member of the family, Src, occurs in advanced colorectal cancer and is associated with a worse outcome. This work tests the hypotheses that Src activation contributes to chemoresistance in some colon tumors and that this resistance can be overcome by use of Src inhibitors. The aims of the proposal were to (1) determine if constitutive Src activation is sufficient to induce oxaliplatin resistance; (2) evaluate the role of reactive oxygen species (ROS) in the activation of Src after oxaliplatin treatment; (3) determine the frequency of Src activation in liver metastases after oxaliplatin treatment; and (4) evaluate the safety, preliminary efficacy, and pharmacodynamics of the combination of dasatinib with oxaliplatin-based therapy in patients with metastatic colorectal cancer. ^ Using a panel of colon cancer cell lines and murine models, I demonstrate that administration of oxaliplatin, a commonly utilized chemotherapy for colorectal cancer, results in an increased activation of Src. The activation occurs acutely in some, but not all, colorectal carcinoma cell lines. Cell lines selected for oxaliplatin resistance are further increased in Src activity. Treatment of cell lines with dasatinib, a non-selective pharmacologic inhibitor of the Src family kinases synergistically killed some, but not all cell lines. Cell lines with the highest acute activation of Src after oxaliplatin administration were the most sensitive to the combination therapy. Previous work demonstrated that siRNA to Src increased sensitivity to oxaliplatin, suggesting that the effects of dasatinib are primarily due to its ability to inhibit Src in these cell lines. ^ To examine the mechanism underlying these results, I examined the effects of reactive oxygen species (ROS), as previous studies have demonstrated that platinum chemotherapeutics result in intracellular oxidative stress. I demonstrated that oxaliplatin-induced reactive oxygen species were higher in the cell lines with Src activation, relative to those in which Src was not activated. This oxaliplatin-induced Src activation was blocked by the administration of anti-oxidants, thereby demonstrating that synergistic killing between dasatinib and oxaliplatin was associated with the ability of the latter to generate ROS. ^ In a murine model of colorectal cancer metastasis to the liver, the combination of dasatinib and oxaliplatin was more effective in reducing tumor volume than either agent alone. However, when oxaliplatin resistant cell lines were treated with a combination of oxaliplatin and AZD0530, an inhibitor in the clinic with increased specificity for Src, no additional benefit was seen, although Src was activated by oxaliplatin and Src substrates were inhibited. The indolent growth of oxaliplatin-resistant cells, unlike the growth of oxaliplatin resistant tumors in patients, precludes definitive interpretation of these results. ^ To further explore Src activation in patients with oxaliplatin exposure and resistance, an immunohistochemistry analysis of tumor tissue from resected liver metastases of colorectal cancer was performed. Utilizing a tissue microarray, staining for phosphorylated Src and FAK demonstrated strong staining of tumor relative to stromal and normal liver. In patients recently exposed to oxaliplatin, there was increased FAK activation, supporting the clinical relevance of the prior preclinical studies. ^ To pursue the potential clinical benefit of the combination of Src inhibition with oxaliplatin, a phase IB clinical trial was completed. Thirty patients with refractory metastatic colorectal cancer were treated with a combination of 5-FU, oxaliplatin, an epidermal-growth factor receptor monoclonal antibody, and dasatinib. The recommended phase II dose of dasatinib was established, and toxicities were quantified. Pharmacodynamic studies demonstrated increased phosphorylation of the Src substrate paxillin after dasatinib therapy. Tumor biopsies were obtained and Src expression levels were quantitated. Clinical benefit was seen with the combination, including a response rate of 20% and disease control rate of 56%, prompting a larger clinical study. ^ In summary, although Src is constitutively activated in metastatic colorectal cancer, administration of oxaliplatin chemotherapy can further increase its activity, through a reactive oxygen species dependent manner. Inhibition of Src in combination with oxaliplatin provides additional benefit in vitro, in preclinical animal models, and in the clinic. Further study of Src inhibition in the clinic and identification of predictive biomarkers of response will be required to further advance this promising therapeutic target. ^

Calcium-dependent mechanisms and long-term potentiation: Role of NMDA receptors, voltage -dependent calcium channels and persistent protein kinase activity

Long-term potentiation (LTP) is a rapidly induced and long lasting increase in synaptic strength and is the leading cellular model for learning and memory in the mammalian brain. LTP was first identified in the hippocampus, a structure implicated in memory formation. LTP induction is dependent on postsynaptic Ca2+ increases mediated by N-methyl-D-aspartate (NMDA) receptors. Activation of other postsynaptic routes of Ca2+ entry, such as voltage-dependent Ca2+ channels (VDCCs) have subsequently been shown to induce a long-lasting increase in synaptic strength. However, it is unknown if VDCC-induced LTP utilized similar cellular mechanisms as the classical NMDA receptor-dependent LTP and if these two forms of LTP display similar properties. This dissertation determines the similarities and differences in VDCC and NMDA receptor-dependent LTP in area CA1 of hippocampal slices and demonstrates that VDCCs and NMDA receptors activate similar cellular mechanisms, such as protein kinases, to induce LTP. However, VDCC and NMDA receptor activated LTP induction mechanisms are compartmentalized in the postsynaptic neuron, such that they do not interact. Consistent with activation properties of NMDA receptors and VDCCs, NMDA receptor and VDCC-dependent LTP have different induction properties. In contrast to NMDA-dependent LTP, VDCC-induced potentiation does not require evoked presynaptic stimulation or display input specificity. These results indicate that there are two different routes of postsynaptic Ca2+ which can induce LTP and the compartmentation of VDCCs and NMDA receptors and/or their resulting Ca2+ increases may account for the distinction between these LTP induction mechanisms.^ One of the molecular targets for postsynaptic Ca2+ that is required for the induction of LTP is protein kinases. Evidence for the role of protein kinase activity in LTP expression is either correlational or controversial. We have utilized a broad range and potent inhibitors of protein kinases to systematically examine the temporal requirement for protein kinases in the induction and expression of LTP. Our results indicate that there is a critical period of persistent protein kinase activity required for LTP induction activated by tetanic stimulation and extending until 20 min after HFS. In addition, our results suggest that protein kinase activity during and immediately after HFS is not sufficient for LTP induction. These results provide evidence for persistent and/or Ca2+ independent protein kinase activity involvement in LTP induction. ^

Regulation of v-Mos kinase activity by phosphorylation

In this thesis, I investigated the effect of cylic AMP-dependent protein kinase (PKA) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of the PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37$\rm\sp{v-mos}$ in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Ser-263 was identified as a residue that is normally phosphorylated at a very low level but whose phosphorylation is dramatically increased upon forskolin treatment. Consistent with the inhibitory role of Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. Based on our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation could be explained at least in part by its inhibition of Mos kinase.^ Combining tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies, I identified Ser-56 as the major in vivo phosphorylation site on v-Mos. I studied the interrelationship between Ser-34 and Ser-56 phosphorylation in regulating v-Mos function. After site-directed mutagenesis to substitute serine residues with alanine or glutamic acid in different combinations to mimick unphosphorylated and phosphorylated serines respectively, various v-Mos mutants were expressed in COS-1 cells. As expected, Ala-34 mutant of v-Mos had very low (less 5% of wild type) kinase activity. The Ala-56 mutant had kinase activity 50% that of wild type. Surprisingly, the Ala-34 Ala-56 double mutant and the Ala-56 mutant exhibited identical kinase activity. On the other hand, Ala-34 Glu-56 double mutant had reduced kinase activity comparable to Ala-34 mutant. These results suggest that the phosphorylation at Ser-56 may serve to inhibit the activation of newly synthesized Mos protein. As predicted from Xenopus c-Mos studies, Glu-34 mutant of v-Mos was highly active (125% that of wild type). Interestingly, consistant with the model involving an inhibitory role of Ser-56 phosphorylation, the Glu-34 Glu-56 double mutant was totally inactive as a kinase. Moreover in my experiments, there was a perfect correlation between the level of v-Mos kinase activity of various mutants and their transforming activity. The latter is dependent upon MEK1 phosphorylation/ activation in v-mos transformed cells. Residues corresponding to both v-Mos Ser-34 and Ser-56 are evolutionarily conserved in c-Mos. Therefore, the cytostatic factor function of c-Mos may be regulated in the same manner as v-Mos kinase activity.^ It has been known that v-mos transforms cells by affecting G1 phase progression of the cell cycle. Here I showed that mos induces cyclin D1 expression in mos transformed NIH 3T3 cells and NRK 6m2 cells, and this induced level was found to be unaffected by serum starvation. Consequently, cyclin D1-Cdk4 and cyclin E-Cdk2 activities increase, and retinoblastoma protein is hyperphosphorylated. Based on studies from several laboratories, these findings suggest that increased amount of cyclin D1-Cdk4 complexes ties up the limited amount of cyclin E-Cdk2 inhibitors (e.g. p27), causing the activation of cyclin E-Cdk2. My results indicate that activation of key cell cycle regulators of G1 phase may be important for cellular transformation by mos. (Abstract shortened by UMI.) ^

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. ^