PubMed · 10502813
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P Collins, C Webb. 1999. Estrogen hits the surface.. https://doi.org/10.1038/13453
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Medicago spp. are able to develop root nodules via symbiotic interaction with Sinorhizobium meliloti. Calcium-dependent protein kinases (CDPKs) are involved in various signalling pathways in plants, and we found that expression of MtCPK3, a CDPK isoform present in roots of the model legume Medicago truncatula, is regulated during the nodulation process. Early inductions were detected 15 min and 3-4 days post-inoculation (dpi). The very early induction of CPK3 messengers was also present in inoculated M. truncatula dmi mutants and in wild-type roots subjected to salt stress, indicating that this rapid response is probably stress-related. In contrast, the later response was concomitant with cortical cell division and the formation of nodule primordia, and was not observed in wild-type roots inoculated with nod (-) strains. This late induction correlated with a change in the subcellular distribution of CDPK activities. Accordingly, an anti-MtCPK3 antibody detected two bands in soluble root extracts and one in the particulate fraction. CPK3::GFP fusions are targeted to the plasma membrane in epidermal onion cells, a localization that depends on myristoylation and palmitoylation sites of the protein, suggesting a dual subcellular localization. MtCPK3 mRNA and protein were also up-regulated by cytokinin treatment, a hormone linked to the regulation of cortical cell division and other nodulation-related responses. An RNAi-CDPK construction was used to silence CPK3 in Agrobacterium rhizogenes-transformed roots. Although no major phenotype was detected in these roots, when infected with rhizobia, the total number of nodules was, on average, twofold higher than in controls. This correlates with the lack of MtCPK3 induction in the inoculated super-nodulator sunn mutant. Our results suggest that CPK3 participates in the regulation of the symbiotic interaction.
Eukaryotic elongation factor-2 kinase (eEF-2 kinase) is a highly conserved calcium/calmodulin-dependent enzyme involved in the regulation of protein translation and cell proliferation. Rapid changes in the activity and abundance of eEF-2 kinase have been observed on growth stimulation, and increased enzyme activity is characteristic of malignant cell growth. Yet the mechanism for controlling the turnover of this kinase is unknown. The ubiquitin-proteasome pathway regulates the degradation of many cellular proteins, including transcription factors, cell cycle regulators, and signal transduction proteins. Therefore, we determined whether the ubiquitin-proteasome pathway regulates the turnover of eEF-2 kinase. We found that eEF-2 kinase was a relatively short-lived protein with a half-life of less than 6 hours. eEF-2 kinase was ubiquitinated in vivo as determined by coimmunoprecipitation and polyubiquitin affinity matrix. Incubation of purified eEF-2 kinase with a source of ubiquitination enzymes (rabbit reticulocyte lysate), purified ubiquitin, and ATP revealed the presence of increasing molecular weight species of ubiquitinated eEF-2 kinase. Treatment of cells with MG132, a proteasome inhibitor, inhibited eEF-2 kinase degradation and induced the accumulation of polyubiquitinated forms of the enzyme, resulting in an increase in its half-life. These results suggest involvement of the proteasome in the turnover of the ubiquitinated kinase. Because eEF-2 kinase is chaperoned by heat shock protein 90 (Hsp90), we next determined if disruption of the Hsp90-eEF-2 kinase complex promoted degradation of the kinase. Treatment of cells with geldanamycin, an Hsp90 inhibitor, enhanced ubiquitination of eEF-2 kinase and decreased the half-life of the kinase to less than 2 hours. These results indicate that cellular levels of eEF-2 kinase are maintained by a balance between association with Hsp90 and degradation by the ubiquitin-proteasome pathway. In conclusion, these data show that the turnover of eEF-2 kinase is regulated by the ubiquitin-proteasome pathway and, therefore, modulating the ubiquitination of eEF-2 kinase might control the abundance of this enzyme and have implications in the treatment of certain forms of cancer.
Calcium/calmodulin-dependent serine protein kinase (CASK) is generally known as a scaffold protein. Here we show that overexpression of CASK resulted in a reduced rate of cell growth, while inhibition of expression of endogenous CASK via RNA-mediated interference resulted in an increased rate of cell growth in ECV304 cells. To explore the molecular mechanism, we identified a novel CASK-interacting protein, inhibitor of differentiation 1 (Id1) with a yeast two-hybrid screening. Furthermore, endogenous CASK and Id1 proteins were co-precipitated from the lysates of ECV304 cells by immunoprecipitation. Mammalian two-hybrid protein-protein interaction assays indicated that CASK possessed a different binding activity for Id1 and its alternative splicing variant. It is known that Id proteins play important roles in regulation of cell proliferation and differentiation. Thus, we speculate that the regulation of cell growth mediated by CASK may be involved in Id1. Our findings indicate a novel function of CASK, the mechanism that remains to be further investigated.