Ca2+/calmodulin-dependent protein kinase type II (CaMK-II) is a multifunctional serine/threonine kinase that is ubiquitously expressed throughout the lifespan of metazoans. Mammals encode four genes (α, β, γ, δ) that generate over forty splice-variants. CaMK-II is important in a myriad of functions, including ion channel regulation, cell-cycle progression, and long term potentiation. In adults, alterations in activation of CaMK-II induce cardiac arrhythmias and heart failure. Developmental roles for CaMK-II are not as well understood since mouse knockouts are embryonic lethal. Therefore the identification of other vertebrate CaMK-II genes will add to our understanding of development. Zebrafish encode seven catalytically active CaMK-II genes (α1, β1, β2, γ1, γ2, δ1, δ2) due to a genome wide duplication event that occurred approximately 250 million years ago. Although, only 20-30% of all duplicated genes were retained, 75% of CaMK-II duplicated genes are transcriptionally active, pointing to a critical role for this signaling protein. mRNA expression patterns demonstrate that CaMK-II is expressed in diverse tissues including retina, pectoral fins, somites, heart, and kidney. Suppression of each gene generates unique phenotypes that mirror the mRNA expression patterns. Of the seven genes, camk2b2 and camk2g1 have the highest maternal contribution in zebrafish, are expressed in mesodermally derived organs, and develop defects similar to human syndromes. In fact, suppression of camk2b2 mimics the phenotype observed in zebrafish mutants of tbx5, the gene mutated in patients with Holt-Oram Syndrome. Camk2g1 morphants also exhibit similar defects as suppression of pkd2, the gene mutated in patients with Autosomal Dominant Polycystic Kidney disease. These roles implicate CaMK-II as an integral protein in the development and maintenance of mesodermally derived tissues.

Calcium (Ca2+)/calmodulin-dependent kinase 2 (CaMK-II) is a multifunctional member of a family of Ca2+/calmodulin-dependent serine/threonine protein kinases that respond to transient intracellular calcium signaling. CaMK-II has been reported to be involved with transcription regulation, cell motility, neuronal development, cell cycle regulation, and more recently early development of vertebrates (Easley et al., 2008; Rothschild et al., 2009; Francescatto et al., 2010). Through previous work in the lab using tandem mass spectrometry and “substrate-trapping mutants”, tumor suppressor protein 53 (p53) was identified as a novel CaMK-II binding partner in tissue culture. In this study, I sought to provide characterization of the functional interaction of p53 and CaMK-II. First, a stable p53 knockdown human cell line (HEK) was established through lentiviral transduction of p53 shRNA and verified with immunoblots and immunostaining assays. Next, the localization of CaMK-II and the cell growth rate in these cells was determined. In wild type HEK cells, catalytically inactive CaMK-II inhibited cell growth, which is consistent with previous studies in mouse fibroblasts with pharmacological inhibition. p53-deficient cells were less sensitive to CaMK-II deficiencies using dominant negative CAMK-II, but not pharmacological disruption. The overall results of this study have provided significant clues to the mechanism between CaMK-II and p53 in the control of cell cycle progression.

Calcium (Ca2+)/calmodulin-dependent kinase 2 (CaMK-II) is one member of a family of Ca2+/calmodulin-dependent protein kinases that responds to intracellular Ca2+ signals (Hudmon, A. and H. Schulman (2002)). CaMK-II is a multifunctional regulator of transcription, cell cycle progression, cell motility and neuronal development. (Wang, C., et al. (2008), Easley, C. A. IV, et al. (2008), Osterhoff, M., et al. (2003), Faison, M. O., et al. (2002)). Recently, CaMK-II has been shown to be important in the early development of vertebrates. In developing zebrafish, disruption of CaMK-II expression has been shown to induce phenotypes similar to those documented in several human diseases. The identification of the tissue-specific binding partners and substrates of CaMK-II which are responsible for specific developmental fates remains a key step in understanding this important protein kinase family. In this thesis research, specific “substrate-trapping” mutants of CaMK-II were designed, introduced into a variety of rodent and human cell lines in culture and used in conjunction with tandem mass spectrometry to identify binding partners, such as β-actin, tropomodulin-3 and Fli-I as well as novel, putative substrates, such as the tumor suppressor protein 53 (p53). This approach was subsequently applied to zebrafish embryos where an overlapping subset of CaMK-II binding proteins to those found in mammalian cell culture were identified. This project represents one of the first studies to identify binding proteins in zebrafish embryos using epitope tagging and mass spectrometry. This research has also established a technical framework for the use of mass spectrometry to characterize the developmental proteome of whole zebrafish embryos or specific zebrafish tissues at any developmental time point.

Ca2+/Calmodulin-dependent protein kinase type II (CaMK-II) is a Serine/Threonine protein kinase that is activated by Ca2+ and Calmodulin to phosphorylate substrates involved in myriad developmental processes. This project implicates CaMK-II in specification of HSCs, and zebrafish provide an ideal embryonic model to study hematopoiesis. Zebrafish genetic manipulation was achieved through: incubation in chemical inhibitors; injection of notochord-targed WT and DN CaMK-II constructs with Transposase; and injection of camk2g1 translation-blocking morpholino antisense oligonucleotide (MO). Whole-mount in situ hybridization (WISH) and immunolocalization on zebrafish embryos allowed visualization of key HSC markers and pathway components that implicated CaMK-II in the specification of HSCs. CaMK-II is a negative regulator of shh expression during HSC specification, but CaMK-II does not influence Shh during its well-documented role in vasculogenesis. CaMK-II appears to affect the spatial distribution of Shh protein, which accumulates near the notochord source and differentially affects expression of Shh target genes based on their distance from the notochord. This project also identifies the specific timing requirement for CaMK-II during HSC specification, as inhibition of CaMK-II consistently reduces HSC specification, but only if administered before 18hpf. CaMK-II also downregulates ezh2 in the DA during the time of HSC specification, and the Ezh2 inhibition rescues the loss of HSCs, suggesting that CaMK-II regulates the secretion of Shh from the notochord to epigenetically regulate expression of key HSC specification genes in the DA through EZH2 methyltransferase.

Previous research showed mutations in muscle sarcoplasmic reticulum-bound calcium handler proteins cause swimming defects in embryonic zebrafish. CaMK-II is a highly conserved Ca2+/calmodulin-dependent protein kinase expressed in all vertebrates has been defined to activate and inactivate multiple Ca2+ handler proteins involved in excitation- contraction coupling and relaxation of cardiac and skeletal muscle. In this study, evidence is provided through pharmacological and genetic intervention that CaMK-II inhibition and overexpression causes swimming defects, particularly response to stimuli and swimming ability, reinforced by immunolocalization of skeletal muscle. Transient CaMK-II inactivation does not have any long-term defects to swimming behavior. Overexpression of wild-type, constitutively active, and dominant-negative CaMK-II-GFP in embryos tended to co-localize in fast muscle which led to defects in swimming behavior. This study concludes that inhibition or overexpression of CaMK-II in skeletal muscle diminishes normal swimming behavior specifically in response to mechanical stimulation and swimming ability.

Ca2+/calmodulin-dependent protein kinase type-II (CaMK-II) is a Ser/Thr protein kinase regulated by Ca2+ and Calmodulin. It is a highly conserved and broadly expressed enzyme and has a unique structure and dynamic regulation. It has the ability to remain active in the absence of Ca 2+ as a result of Ca2+ dependent autophosphorylation. CaMK-II phospliorylates proteins involved in neurotransmitter secretion, long term potentiation, cytoskeletal dynamics, gene transcription, and cell motility. To support existing and identify new intracellular roles of CaMK-II, potential binding partners were identified. This was accomplished by transfecting and purifying "FLAG-tagged" CaMK-II's (α, βE, δC, and δE). CaMK-II associated proteins were then identified using tandem mass spectrometry. Known binding partners were identified using this approach, including CaMK-II and calmodulin, verifying the approach's validity. Additionally several unexpected but interesting proteins were identified, including the gelsolin related actin binding protein, Flightless-I. Fli-I is an actin binding and capping protein that also functions as a transcriptional coactivator. The CaMK-II-Fli-I interaction was confirmed with endogenous (un-tagged) proteins. The association and localization of Fli-I are dependent on CaMK-II's activity state, although Fli-I is not a substrate of CaMK-II. When CaMK-II is inhibited, Fli-I translocates to the nucleus. Conversely when CaMK-II is artificially activated using a Ca2+ ionophore, Fli-I returns to the cytosol. The discovery of this reversible interaction epresents a potentially new CaMK-II regulated pathway and likely serves as a link between Ca2+ based signal transduction pathways and regulation of the actin component of the cytoskeleton and transcription.

Transient elevations in Ca2+ have previously been shown to promote focal adhesion disassembly and cell motility. Yet the targets of these Ca2+ transients have not been fully examined. In this study, we demonstrate that CaMK-II, a Ca2+/calmodulin dependent protein kinase, is activated in response to β1 integrin engagement with fibronectin to influence fibroblast adhesion and motility. We also show that CaMK-II is dynamically localized to the cell surface using Total Internal Reflection Fluorescence microscopy (TIRFm) and that inhibition of CaMK-II with two mechanistically distinct, membrane permeant inhibitors accelerates spreading on fibronectin, enlarges paxillin-containing focal adhesions and blocks cell motility. On the other hand, expression of constitutively active CaMK-II reduces cell attachment, eliminates paxillin from focal adhesions and decreases the phospho-tyrosine levels of both FAK and paxillin. Cell spreading, paxillin incorporation into focal adhesions and phospho-tyrosine levels of FAK and paxillin are restored when cells expressing constitutively active CaMK-II are subsequently treated with myr-AIP, a specific CaMK-II catalytic inhibitor. Like CaMK-II inhibition, constitutively active CaMK-II blocks cell motility. Thus, both CaMK-II inhibition and constitutive activation block cell motility through over-stabilization or destabilization of focal adhesions, respectively. These findings provide the first direct evidence that CaMK-II promotes focal adhesion turnover and thus enables cell motility by stimulating tyrosine dephosphorylation of focal adhesion proteins.

Coordinated inductive and morphogenetic processes of gastrulation establish the zebrafish body plan. Gastrulation includes massive cell rearrangements to generate the three germ layers and shape the embryonic body. Three modes of cell migration must occur during vertebrate gastrulation and include: epiboly, internalization of the presumptive mesendoderm and convergent extension (C&E). C&E movements narrow the germ layers mediolaterally (convergence) and elongate them anteroposteriorly (extension) to define the embryonic axis. The molecular mechanisms regulating coordinated cell migrations remain poorly understand and studying these has become of great interest to researchers. Understanding cell migration during development is highly relevant to a number of human physiological processes. Abnormal cell migration during early development can lead to congenital defects, with improper cell migration during adult life potentially leading to the invasion and metastasis of cancer. By studying cell migration events, in vivo, new insights are to be found to both the function and malfunction of key embryonic and postembryonic migratory events. The non-canonical Wnt pathway has been identified as an evolutionarily conserved signaling pathway, regulating C&E cell movements during vertebrate gastrulation. With the absence of the non-canonical Wnts (ncWnts), Wnt5 and Wnt11, during zebrafish development leading to a shorter and broader body axis with defects in elongation during segmentation resulting in undulation of the notochord. While it is clear ncWnts are necessary for C&E, many of the downstream effectors regulating these cell movements have not been defined. Previous research has shown that activation of ncWnt signaling through Wnt5 or Wnt11 results in an increase in intracellular Ca2+ during zebrafish gastrulation. To determine if the Ca2+/Calmodulin-dependent protein kinase, CaMK-II, is a potential downstream target of the Ca2+ increases during ncWnt activation, CaMK-II’s role in C&E was assessed. This study identifies camk2b1 and camk2g1 as being necessary for C&E movements, and outlines the phenotype of the overall embryo as well as individual cells of camk2b1 and camk2g1 morphants. The defects of CaMK-II morphants are specifically linked to alterations in C&E cell movements, while cell fate and proliferation are unaffected. An increase in CaMK-II activation during gastrulation produces similar C&E defects, demonstrating the specificity of CaMK-II’s activation in facilitating these highly coordinated cellular movements. We show that CaMK-II is working downstream Wnt 11 and in parallel to JNK signaling during gastrulation C&E. Overall, these data identify CaMK-II as a required component of C&E movements during zebrafish development, downstream ncWnt signaling, and altering cell migration through changes in cell shape

A fecundação resulta no aumento intracelular de cálcio que é necessário para a transição do oócito até o estádio de zigoto. Os eventos que ocorrem durante esta transição são caracterizados como ativação, sendo estes dependentes de cálcio. Entretanto, os eventos bioquímicos que ocorrem durante a ativação ainda não estão completamente elucidados. A proteína quinase C (PKC) e a proteína quinase dependente de cálcio/calmodulina (CaMKII), por apresentarem atividade durante a fecundação e por serem ativadas por cálcio são implicadas na regulação dos eventos da ativação. Entretanto, existem muitas dúvidas sobre o real papel destas proteínas na ativação do oócito. Deste modo, o objetivo do presente trabalho foi avaliar o papel da PKC e da CaMKII na ativação de oócitos bovinos. Para tal, oócitos bovinos maturados in vitro foram ativados partenogeneticamente (AP) com cálcio ionóforo A23187 (5μM) por 5 minutos, sendo a retomada da meiose, a organização do citoesqueleto e do retículo endoplasmático (RE) avaliada 1 hora após a ativação. No experimento 1 foi avaliado o papel da CaMKII nestes eventos. Os oócitos foram AP na presença ou ausência de 100M do inibidor de CaMKII (Autocamtide-2 Related Inhibitory Peptide, Myristoylated). A inibição da CaMKII não afetou a retomada da meiose e nem a distribuição dos RE, após a AP. Entretanto, não ocorreu a rotação do fuso meiótico no estádio de telófase II quando a CaMKII foi inibidada. Estes resultados demonstram que embora a CaMKII não tenha efeito na retomada da meiose, esta proteína participa na progressão do ciclo celular de oócitos bovinos, após a AP. No experimento 2 foi avaliado o papel da PKC em oócitos bovinos AP. Os oócitos foram ativados partenogeneticamente na presença ou ausência de 10μM do inibidor de PKC (Bisindolymaleimide I). A inibição da PKC não afetou a retomada da meiose e nem a progressão pelo ciclo celular até o estádio de telófase II. Entretanto, a organização do RE foi afetada pela inibição da PKC. Resultado semelhante foi obtido quando os oócitos foram ativados na presença de citocalasina C, um despolimerizador de filamentos de actina. O presente experimento demonstra a participação da via PKC-actina na organização do RE na ativação de oócitos bovinos. / The intracellular calcium increase resulting from fertilization is necessary for oocyte transition to zygote. The events that occur during this transition are characterized as activation, which are dependent on calcium. However the biochemical events that occur during this activation are still not fully elucidated. The protein kinase C (PKC) and the calcium/calmodulin-dependent protein kinase II (CaMKII), are involved in regulating the events of activation, since these proteins have activity during fertilization and are activated by calcium. However there are many doubts about the real role of these proteins in the oocyte activation. Thus, the objective of this study was to evaluate the role of PKC and CaMKII in bovine oocyte activation. For this purpose, in vitro matured bovines oocytes were parthenogenetically activated (PA) by using calcium ionophore A23187 (5μM) for five minutes, and the resumption of meiosis, the cytoskeleton organization and the endoplasmic reticulum (ER) organization were evaluated 1 hour post-activation. In experiment 1, were evaluated the role of CaMKII in these events. The oocytes were PA in the presence or absence of 100M of CaMKII inhibitor (Autocamtide-2 Related Inhibitory Peptide, Myristoylated). The inhibition of CaMKII did not affect the meiosis resumption and the ER after the PA. However, there was no spindle rotation at telophase II stage when the CaMKII was inhibited. These results showed that although the CamKII has no effect on resumption of meiosis, it participates in the regulation of cell cycle progression after PA of bovine oocytes. In experiment 2, was evaluated the role of PKC on PA bovine oocytes. The oocytes were parthenogenetically activated in the presence or absence of 10μM of PKC inhibitor (Bisindolymaleimide I). The PKC inhibition did not affected the resumption of meiosis and the progression through the cell cycle until the stage of telophase II. However, the ER organization was affected by PKC inhibition. A similar result was obtained when the oocytes were activated in the presence of cytochalasin C, which promotes the depolymerization of the actin filaments. The current experiment showed the participation of the PKC-actin pathway at the ER organization in the bovine oocytes activation.

A fecundação resulta no aumento intracelular de cálcio que é necessário para a transição do oócito até o estádio de zigoto. Os eventos que ocorrem durante esta transição são caracterizados como ativação, sendo estes dependentes de cálcio. Entretanto, os eventos bioquímicos que ocorrem durante a ativação ainda não estão completamente elucidados. A proteína quinase C (PKC) e a proteína quinase dependente de cálcio/calmodulina (CaMKII), por apresentarem atividade durante a fecundação e por serem ativadas por cálcio são implicadas na regulação dos eventos da ativação. Entretanto, existem muitas dúvidas sobre o real papel destas proteínas na ativação do oócito. Deste modo, o objetivo do presente trabalho foi avaliar o papel da PKC e da CaMKII na ativação de oócitos bovinos. Para tal, oócitos bovinos maturados in vitro foram ativados partenogeneticamente (AP) com cálcio ionóforo A23187 (5μM) por 5 minutos, sendo a retomada da meiose, a organização do citoesqueleto e do retículo endoplasmático (RE) avaliada 1 hora após a ativação. No experimento 1 foi avaliado o papel da CaMKII nestes eventos. Os oócitos foram AP na presença ou ausência de 100M do inibidor de CaMKII (Autocamtide-2 Related Inhibitory Peptide, Myristoylated). A inibição da CaMKII não afetou a retomada da meiose e nem a distribuição dos RE, após a AP. Entretanto, não ocorreu a rotação do fuso meiótico no estádio de telófase II quando a CaMKII foi inibidada. Estes resultados demonstram que embora a CaMKII não tenha efeito na retomada da meiose, esta proteína participa na progressão do ciclo celular de oócitos bovinos, após a AP. No experimento 2 foi avaliado o papel da PKC em oócitos bovinos AP. Os oócitos foram ativados partenogeneticamente na presença ou ausência de 10μM do inibidor de PKC (Bisindolymaleimide I). A inibição da PKC não afetou a retomada da meiose e nem a progressão pelo ciclo celular até o estádio de telófase II. Entretanto, a organização do RE foi afetada pela inibição da PKC. Resultado semelhante foi obtido quando os oócitos foram ativados na presença de citocalasina C, um despolimerizador de filamentos de actina. O presente experimento demonstra a participação da via PKC-actina na organização do RE na ativação de oócitos bovinos. / The intracellular calcium increase resulting from fertilization is necessary for oocyte transition to zygote. The events that occur during this transition are characterized as activation, which are dependent on calcium. However the biochemical events that occur during this activation are still not fully elucidated. The protein kinase C (PKC) and the calcium/calmodulin-dependent protein kinase II (CaMKII), are involved in regulating the events of activation, since these proteins have activity during fertilization and are activated by calcium. However there are many doubts about the real role of these proteins in the oocyte activation. Thus, the objective of this study was to evaluate the role of PKC and CaMKII in bovine oocyte activation. For this purpose, in vitro matured bovines oocytes were parthenogenetically activated (PA) by using calcium ionophore A23187 (5μM) for five minutes, and the resumption of meiosis, the cytoskeleton organization and the endoplasmic reticulum (ER) organization were evaluated 1 hour post-activation. In experiment 1, were evaluated the role of CaMKII in these events. The oocytes were PA in the presence or absence of 100M of CaMKII inhibitor (Autocamtide-2 Related Inhibitory Peptide, Myristoylated). The inhibition of CaMKII did not affect the meiosis resumption and the ER after the PA. However, there was no spindle rotation at telophase II stage when the CaMKII was inhibited. These results showed that although the CamKII has no effect on resumption of meiosis, it participates in the regulation of cell cycle progression after PA of bovine oocytes. In experiment 2, was evaluated the role of PKC on PA bovine oocytes. The oocytes were parthenogenetically activated in the presence or absence of 10μM of PKC inhibitor (Bisindolymaleimide I). The PKC inhibition did not affected the resumption of meiosis and the progression through the cell cycle until the stage of telophase II. However, the ER organization was affected by PKC inhibition. A similar result was obtained when the oocytes were activated in the presence of cytochalasin C, which promotes the depolymerization of the actin filaments. The current experiment showed the participation of the PKC-actin pathway at the ER organization in the bovine oocytes activation.