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J Biol Chem,
1999]
Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK alpha and beta shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP- domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP- domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.
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J Biol Chem,
1999]
We have recently demonstrated that Caenorhabditis elegans Ca(2+)/calmodulin-dependent protein kinase kinase (CeCaM-KK) can activate mammalian CaM-kinase IV in vitro (Tokumitsu, H., Takahashi, N., Eto, K., Yano, S., Soderling, T.R., and Muramatsu, M. (1999) J. Biol. Chem. 274, 15803-15810). In the present study, we have identified and cloned a target CaM-kinase for CaM-KK in C. elegans, CeCaM-kinase I (CeCaM-KI), which has approximately 60% identity to mammalian CaM-KI. CeCaM-KI has 348 amino acid residues with an apparent molecular mass of 40 kDa, which is activated by CeCaM-KK through phosphorylation of Thr(179) in a Ca(2+)/CaM-dependent manner, resulting in a 30-fold decrease in the K(m) of CeCaM-KI for its peptide substrate. Unlike mammalian CaM-KI, CeCaM-KI is mainly localized in the nucleus of transfected cells because the NH(2)-terminal six residues ((2)PLFKRR(7)) contain a functional nuclear localization signal. We have also demonstrated that CeCaM-KK and CeCaM-KI reconstituted a signaling pathway that mediates Ca(2+)-dependent phosphorylation of cAMP response element-binding protein (CREB) and CRE-dependent transcriptional activation in transfected cells, consistent with nuclear localization of CeCaM-KI. These results suggest that the CaM-KK/CaM-KI cascade is conserved in C. elegans and is functionally operated both in vitro and in intact cells, and it may be involved in Ca(2+)-dependent nuclear events such as transcriptional activation through phosphorylation of CREB.
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Parasitol Today,
1990]
Onchocerca volvulus causes a disease of significant socio-economic importance in West Africa, the Arabian Peninsula and regions of South America. Brian Duke explains that, despite the advent of ivermectin, the prospects for the eradication of this potentially highly debilitating infection are remote. Moreover, the logistical problems associated with the control of morbidity caused by the parasite are considerable, and are highly dependent on the ability to sustain financial support and political will over many decades.
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Proc Natl Acad Sci U S A,
2001]
We have constructed DNA microarrays containing 17,871 genes, representing about 94% of the 18,967 genes currently annotated in the Caenorhabditis elegans genome. These DNA microarrays can be used as a tool to define a nearly complete molecular profile of gene expression levels associated with different developmental stages, growth conditions, or worm strains. Here, we used these full-genome DNA microarrays to show the relative levels of gene expression for nearly every gene during development, from eggs through adulthood. These expression data can help reveal when a gene may act during development. We also compared gene expression in males to that of hermaphrodites and found a total of 2,171 sex-regulated genes (P < 0.05). The sex-regulated genes provide a global view of the differences between the sexes at a molecular level and identify many genes likely to be involved in sex-specific differentiation and behavior.
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Dev Biol,
2025]
How life emerged, how species diversify and interact, how cells work individually and collectively, and how a single cell gives rise to the trillions that constitute us all, are some of life's most profound questions. For the last 20 years, I've been a professor in the Biology Department at Duke University and spent summers teaching at the Marine Biological Laboratory in Woods Hole. I use the small nematode worm C. elegans in my research to understand cell-extracellular matrix interactions, and I've taught and mentored hundreds of undergraduate students, graduate students, postdocs and many junior faculty. Young scientists often become discouraged during their training-the modest pay, the long hours, and the herculean task and many setbacks that occur conducting research, writing grants and papers, teaching, and managing a lab. It can seem to be an impossible challenge and leads many young scientists to curtail their training and leave fulfilling scientific careers. A core element to success is that scientific skills and expertise evolve slowly over an entire career through engagement-learning by doing-and requires patience, acceptance of failures, continual exploration, and a flexible growth mindset. Everyone also takes a unique path and there is no simple rulebook. This is difficult to convey through courses and workshops. By sharing some tools and approaches I've learned through my own setbacks, observations, and reading, I hope to help junior scientists view challenges in this career more as lifelong opportunities for building new skillsets and growth, much like the emergence of form and function that occurs during developmental morphogenesis. Embracing constant change and evolution can lead to a deeply meaningful and fulfilling career.