A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, M J Fisher, M Tabish, R A Clegg, H H Rees

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

L C Bowen et al. (2002) European Worm Meeting "The C. elegans cyclic AMP-dependent protein kinase (PK-A) catalytic subunit gene (kin-1)"

L C Bowen, M Tabish, M J Fisher, H H Rees, R A Clegg

[European Worm Meeting, 2002]

PK-A is one of the major multifunctional ser/thr protein kinases found in eukaryotic organisms. The holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. Both R and C subunits occur in multiple isoforms encoded by distinct genes. In many organisms, the genes that encode the C subunits also show alternative splicing behaviour. This generates an even broader array of C subunit heterogeneity. The diversity of C subunit polypeptides may enable this protein kinase to selectively target substrate polypeptides for phosphorylation.

Martyna W Pastok et al. (2007) International Worm Meeting "Studies on the C. elegans Protein Kinase A (PK-A) Catalytic and Regulatory Subunit Genes."

Huw H Rees, Martyna W Pastok, Michael J Fisher, Caroline Dart, Patrica A Murray

[International Worm Meeting, 2007]

The cyclic AMP-dependent protein kinase (protein kinase A; PK-A) holoenzyme consists of two catalytic (C) subunits bound to two regulatory (R) subunits, forming an inactive heterotetramer (R2C2). Binding of cyclic AMP to the R subunits results in the dissociation of catalytically active monomeric C subunits. These C subunits catalyse the protein phosphorylation events underlying the diverse range of cellular processes triggered by alterations in cyclic AMP concentration. In C. elegans, C and R subunits are encoded by the kin-1 and kin-2 genes respectively. We have undertaken both bioinformatic and molecular studies on the structure and expression of these genes, in C. elegans. We have also undertaken comparative bioinformatic studies on orthologus genes, and gene products, in C. briggsae and C. remanei. These studies have highlighted the potential significance of alternative splicing processes, as a means of generating multiple isoforms of both the C and R subunits in C. elegans. The possible significance of such multiple isoforms is discussed in the context of the subcellular distribution of PK-A and in relation to the targeting of specific phosphorylation events, in responses to changes in intracellular cyclic AMP concentration.

Shimizu M et al. (1998) Midwest Worm Meeting "MOLECULAR INTERACTION OF MYOTONIC DYSTROPHY PROTEIN KINASE: THE STRUCTURE AND FUNCTION OF DMPK FAMILY MEMBERS IN C. ELEGANS"

Shimizu M, Epstein HF

[Midwest Worm Meeting, 1998]

Myotonic dystrophy (DM) is the most common form of muscular dystrophy in adults. DM is inherited as an autosomal dominant disorder with complex clinical findings which can include skeletal muscle weakness and myotonia, cardiac arrhythmias, characteristic cataracts of the eye lens, testicular atrophy, and insulin-resistant diabetes. This complex phenotype is closely associated with an unstable expansion of a CTG repeat in the 3'-UTR region of a locus encoding DM protein kinase (DMPK). DMPK is a Ser/Thr protein kinase, which is confirmed by enzymological studies of recombinant DMPK. It represents a new group of proteins called "the DMPK family". We are interested in the molecular interactions of DMPK in several model systems, including the structure and function of DMPK family members in C. elegans. Because many basic biological pathways are conserved in nature (e.g. Ras, TGF-b, Notch pathways, etc.), a DMPK family member in the nematode C. elegans was chosen. The scientific knowledge available in the genetics and cytology of C. elegans will facilitate the dissection of possible pathways of DMPK interactions. DMPK (629aa) has four domains; N-terminal Leucine-rich repeat (L), Ser/Thr protein kinase (PK), coild-coil zipper (H), and C-terminal transmembrane domain (T). So far there is no DMPK ortholog in C. elegans although a couple of PK homologs exist. The one we chose as the best homolog for DMPK is a predicted gene in cosmid K08B12 on chromosome V and we call it as CeDMPK. CEDMPK has 53.5% identity in its catalytic domain to human DMPK. The predicted protein (172kDa) is about 2.5 times larger than human DMPK (70kDa). In addition to its DMPK-like Ser/Thr kinase domain, CeDMPK is predicted to contain a large coiled-coil region, a cysteine-rich zinc-finger domain (CRD), a pleckstrin-homology motif (PH), and a Cdc42/Rac binding domain (CRIB). CEDMPK share the homology with human DMPK not only at PK domain but also N-terminal hydrophobic helical region and a part of coiled-coil domain. We analyzed the CeDMPK expression pattern with a GFP-fusion construct. We found the pharyngeal muscle expression at all developmental stages from late embryo to adult, some neurons in the head and tail (also ventral nerve code) in L1, body-wall muscle in L2-L3, and vulva in L4. Potential knockout phenotypes obtained by RNA-mediated inhibition will also be discussed.

Henss, Thilo et al. (2021) International Worm Meeting "Optogenetic tools for manipulation of cyclic nucleotides, functionally coupled to CNG-channels"

Nagel, Georg, Henss, Thilo, Hirschhauser, Alexander, Scheib, Ulrike, Schneider-Warme, Franziska, Hegemann, Peter, Gao, Shiqiang, Gottschalk, Alexander, Pieragnolo, Alessia, Nagpal, Jatin

[International Worm Meeting, 2021]

The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers that regulate numerous biological processes by activating e.g. protein kinases (PKA and PKG) or cyclic nucleotide gated channels (CNGCs). In eukaryotic GPCR signalling, cAMP is generated predominantly by membrane-bound adenylyl cyclases (mbACs), which are located in microdomains together with GPCRs, PK(A) and their targets. The existing optogenetic toolbox in C. elegans is restricted to soluble adenylyl cyclases (i.e. microbial photoactivatable adenylyl cyclases (PACs) from Euglena (euPAC) and Beggiatoa (bPAC), and the synthetic phytochrome-linked cyclases IlaC22 k27 and PaaC), the membrane-bound Blastocladiella emersonii guanylyl cyclase opsin (BeCyclOp) and hyperpolarising rhodopsins (e.g. Natronomonas pharaonis halorhodopsin - NpHR). Yet missing are membrane-bound photoactivatable adenylyl cyclases (mbPACs) and hyperpolarizers based on K+-currents. To obtain mbPACs, we mutated 2-3 key amino acids in the active site of Blastocladiella and Catenaria CyclOps, which are particular in combining a rhodopsin and a guanylyl cyclase domain. For characterization of photoactivatable nucleotidyl cyclases, we expressed the proteins alone or in combination with CNGCs ("two-component optogenetics") in muscle cells and cholinergic motor neurons. To investigate the extent of optogenetic cNMP production and the ability of the systems to de- or hyperpolarise cells, we performed behavioural analyses (locomotion, muscle contraction), measured cNMP content in vitro, and compared in vivo expression levels. We implemented Catenaria CyclOp as a new tool for cGMP production, allowing fine-control of cGMP levels. We established the mbPACs YFP-BeCyclOp(A-2x) and YFP-CaCyclOp(A-2x), based on mutated versions ("A-2x") of Be and Ca CyclOp, enabling more efficient and specific cAMP signalling compared to soluble bPAC, despite lower overall cAMP production. For hyperpolarization of excitable cells by two-component optogenetics, we introduced the cAMP-gated K+-channel SthK from Spirochaeta thermophila and combined it with bPAC, BeCyclOp(A-2x), or YFP-BeCyclOp(A-2x). As an alternative, we implemented the Blastocladiella emersonii cGMP-gated K+-channel BeCNG1 together with BeCyclOp. In summary, we established a comprehensive suite of optogenetic tools for cNMP manipulation, useful for applications in many cell types, including sensory neurons, and for potent hyperpolarization.

Land M et al. (1991) International C. elegans Meeting "CHARACTERIZATION OF cDNAs ENCODING TWO PROTEIN KINASE C (PKC) ISOFORMS AND A RELATED, BUT NOVEL PK IN C. ELEGANS."

Land M, Rubin CS, Islas-Trejo A

[International C. elegans Meeting, 1991]

The Ca2+/diacyglycerol (DAG) pathway for transmembrane signaling is highly conserved in eukaryotes. Extracellular stimuli (e.g., growth factors) activate phospholipase C, which catalyzes the generation of DAG and inositol triphosphate (IP3) from membrane phospholipids. DAG and Ca2+ mobilized by IP3 are co-activators of the classical isoforms of PKC, which amplify and propagate physiological signals by phosphorylating substrate proteins at regulatory sites. The phorbol ester TPA binds to and constitutively activates PKC, by mimicking DAG. TPA causes uncoordinated movement and reproductive defects in C. elegans. Miwa et.al. (Science, 243:1713-1716, 1989), have demonstrated that C. elegans mutants that are resistant to TPA, have defects in the tPa-l gene which encodes a PKC isoform. To study further PKC isoforms and PKC-related enzymes in C. elegans a cDNA library was screened with a PKC-specific oligonucleotide. cDNAs encoding 2 PKC isoforms and a novel Ser PK (PKL) were cloned and characterized. Both PKCs have conserved N-terminal Cys repeats and Cterminal catalytic domains. PKCl appears to be Ca2+independent and is related to the CNS-specific PKC1 of mammals. PKCl cDNA hybridizes with 2 mRNAs (2.3,2.8 kb). Considerable heterogeneity is evident at the 5'-ends of PKCl cDNAs; PKCl transcripts that are either trans-spliced with SLl, abortively (?) spliced or contain a 160 bp ORF 5' extension (due to cis-splicing) have been discovered. Multiple PKCl mRNAs and N-termini may reflect cell- or developmental stage-specific regulatory processes. PKC2 appears to be a Ca2+/DAG stimulated enzyme that is encoded by two mRNAs (2.0,2.6 kb). PKCl and PKC2 were mapped to LG3 and LGX, respectively, whereas tpa-l is located on LG4. PKL is a 166 kDa protein encoded by a 5.2 kb mRNA that is transspliced with SLl. Its centrally located catalytic domain is 36% identical to the corresponding domains of PKCs. The C-terminus of PKL is highly basic, contains 3 phosphorylation sites for PKA, and is not homologous with other proteins. The content of PKL mRNA declines 67% in TPA-treated wild-type C. elegans; no change in PKL mRNA is observed when tpa-l nematodes are exposed to TPA.

Mingfu Wu et al. (2003) International Worm Meeting "Components of the Planar Cell Polarity pathway function in the control of B cell polarity in C. elegans"

Mingfu Wu, Michael A Herman

[International Worm Meeting, 2003]

During development, different cell fates are generated by asymmetric cell divisions and cell-cell interactions. The orientation of these cells to the body axis of an animal gives each cell a polarity. We are interested in how asymmetries are generated and how cell polarity is controlled during metazoan development. In some cases, the asymmetry of the daughter cells generated by an asymmetric division is apparent as a difference in cell size. The C. elegans male specific blast cell, B, divides asymmetrically to generate a large anterior-dorsal daughter (B.a) and a smaller posterior-ventral daughter (B.p). lin-44/Wnt and lin-17/Fz are involved in the control of B cell polarity. Mutations in lin-44 cause a reversal of B cell polarity, while mutations in lin-17 cause a symmetric B cell division. lin-44 and lin-17 also control the polarity of T cell, causing the reversal and loss of T cell polarity, respectively. A non-canonical Wnt pathway involved in planar cell polarity (PCP) has been described in both Drosophila and vertebrate systems. In Drosophila, epithelial cells in the eye, wing and thorax become polarized within the epithelial sheet by the action of the PCP pathway. The six PCP core genes, Fz, Dsh, Fmi, Pk, Stbm and Dgo, have been identified in Drosophila and their function and asymmetric distribution in the wing cells have been characterized. In addition, the PCP pathway plays an important role in the regulation of convergent extension movements during gastrulation in both zebrafish and Xenopus embryos. We have identified C. elegans PCP pathway homologs and used RNAi as well as available mutants to disrupt their functions to determine whether they are involved in the control of B cell polarity. We have found that disruption of lin-44/Wnt, lin-17/Fz, mig-5/Dsh, unc-44/Dgo, rho-1/RhoA, let-502/Rock and POP-1/Tcf functions cause B cell polarity defects, but with the exception of lin-44 and lin-17, do not cause T cell polarity defects. We have used double mutants analysis to determine that rho-1 functions downstream of lin-44. Our results suggest that components of the conserved PCP/Wnt pathway are involved in the control of B cell polarity. We are also performing a genetic screen for mutants that affect B cell polarity. We have already isolated one mutant, mh52, which displays a high degree of B cell polarity reversals, but does not affect T cell polarity. mh52 complements lin-44 and lin-17 mutants, suggesting that mh52 defines a new gene involved in the control of B cell polarity. This is only the second gene that causes highly penetrant B cell reversals. Our long term goal is to determine how the new genes we identify function with the known PCP genes to control B cell polarity.