Genome-Wide Analysis of the Apple CBL Family Reveals That Mdcbl10.1 Functions Positively in Modulating Apple Salt Tolerance

Abiotic stresses are increasingly harmful to crop yield and quality. Calcium and its signaling pathway play an important role in modulating plant stress tolerance. As specific Ca2+ sensors, calcineurin B-like (CBL) proteins play vital roles in plant stress response and calcium signaling. The CBL family has been identified in many plant species; however, the characterization of the CBL family and the functional study of apple MdCBL proteins in salt response have yet to be conducted in apple. In this study, 11 MdCBL genes were identified from the apple genome. The coding sequences of these MdCBL genes were cloned, and the gene structure and conserved motifs were analyzed in detail. The phylogenetic analysis indicated that these MdCBL proteins could be divided into four groups. The functional identification in Na+-sensitive yeast mutant showed that the overexpression of seven MdCBL genes could confer enhanced salt stress resistance in transgenic yeast. The function of MdCBL10.1 in regulating salt tolerance was also verified in cisgenic apple calli and apple plants. These results provided valuable insights for future research examining the function and mechanism of CBL proteins in regulating apple salt tolerance.

Molecular characterisation and expression profiling of calcineurin B-like (CBL) genes in Chinese cabbage under abiotic stresses

Calcium signals act as a second messenger in plant responses to various abiotic stresses, which regulate a range of physiological processes. Calcium-binding proteins, like calcineurin B-like (CBL) proteins, belong to a unique group of calcium sensors that play a role in calcium signalling. However, their identities and functions are unknown in Chinese cabbage. In this study, 17 CBL genes were identified from the Brassica rapa L. (Chinese cabbage) database and Br135K microarray datasets. They were used to construct a phylogenetic tree with known CBL proteins of other species. Analysis of genomic distribution and evolution revealed different gene duplication in Chinese cabbage compared to Arabidopsis. The microarray expression analysis showed differential expression of BrCBL genes at various temperatures. Organ-specific expression was observed by RT–PCR, and qRT–PCR analyses revealed responsiveness of BrCBL genes to cold, drought and salt stresses. Our findings confirm that CBL genes are involved in calcium signalling and regulate responses to environmental stimuli, suggesting this family gene have crucial role to play in plant responses to abiotic stresses. The results facilitate selection of candidate genes for further functional characterisation. In addition, abiotic stress-responsive genes reported in this study might be exploited for marker-aided backcrossing of Chinese cabbage.

The Tyrosine Kinase-Binding and Proline-Rich Domains of Mutant CBL Are Essential for Leukemogenesis

Mutations of the tyrosine kinase-directed ubiquitin ligase CBL are associated with myeloid malignancies, yet the molecular mechanisms by which this tumor suppressor becomes a dominant oncogene to initiate the leukemogenic process are unclear. In this study, we used systematic mutagenesis to delineate the importance of the various protein-protein interaction domains/motifs of a CBL mutant that is most frequently found in human leukemias, Y371H, in inducing leukemogenesis. We tested the impact of these secondary mutations on the ability of CBL-Y371H to impart hypersensitivity to cytokines and to upregulate the associated signaling pathways in the TF1 leukemia cell line model, and in mouse hematopoietic stem/progenitor cells from Cbl-null mice to mimic the lack of wildtype CBL expression in most of the mutant CBL bearing leukemia patients. The secondary mutations included: Cbl G306E, to abrogate the ability of the TKB domain of mutant Cbl to bind to activated tyrosine kinases; internal deletion of the proline-rich domain (AA 477-688) to abrogate interactions with SH3 domain containing partners; Cbl-Y700/731/774F triple phosphorylation site mutant predicted not to interact with the SH2 domain-containing partners; and Cbl-1-436 deletion construct lacking all C-terminal motifs. Analyses of stably expressed mutants in TF-1 cells for hypersensitivity to SCF1 demonstrated an essential role of an intact TKB domain and a particularly important role of the proline-rich domain. Transient retroviral expression in Cbl-null primary murine hematopoietic stem/progenitor cells confirmed these results. Deletion of the proline-rich domain led to substantially less tyrosine phosphorylation of the oncogenic mutant Cbl as well as of the c-Kit receptor upon SCF stimulation, and cells expressing this mutant lacked the sustained activation of Erk1/2 and Akt typically seen after SCF stimulation of the Cbl-Y371H-expressing cells. Together, our data provide conclusive evidence that interaction of leukemogenic mutant Cbl proteins with the upstream tyrosine kinase (via the TKB domain) and with partner proteins (via the proline-rich domain) provides a basic mechanism for gain of function phenotype of mutant Cbl proteins. Our studies suggest that identification of the leukemogenesis-critical partners of the proline-rich domain and targeting of the TKB-tyrosine kinase interface provide new therapeutic approaches against mutant Cbl-driven leukemias. Disclosures No relevant conflicts of interest to declare.

Tonoplast CBL–CIPK calcium signaling network regulates magnesium homeostasis in Arabidopsis

Although Mg2+ is essential for a myriad of cellular processes, high levels of Mg2+ in the environment, such as those found in serpentine soils, become toxic to plants. In this study, we identified two calcineurin B-like (CBL) proteins, CBL2 and CBL3, as key regulators for plant growth under high-Mg conditions. The Arabidopsis mutant lacking both CBL2 and CBL3 displayed severe growth retardation in the presence of excess Mg2+, implying elevated Mg2+ toxicity in these plants. Unexpectedly, the cbl2 cbl3 mutant plants retained lower Mg content than wild-type plants under either normal or high-Mg conditions, suggesting that CBL2 and CBL3 may be required for vacuolar Mg2+ sequestration. Indeed, patch-clamp analysis showed that the cbl2 cbl3 mutant exhibited reduced Mg2+ influx into the vacuole. We further identified four CBL-interacting protein kinases (CIPKs), CIPK3, -9, -23, and -26, as functionally overlapping components downstream of CBL2/3 in the signaling pathway that facilitates Mg2+ homeostasis. The cipk3 cipk9 cipk23 cipk26 quadruple mutant, like the cbl2 cbl3 double mutant, was hypersensitive to high-Mg conditions; furthermore, CIPK3/9/23/26 physically interacted with CBL2/3 at the vacuolar membrane. Our results thus provide evidence that CBL2/3 and CIPK3/9/23/26 constitute a multivalent interacting network that regulates the vacuolar sequestration of Mg2+, thereby protecting plants from Mg2+ toxicity.

The Role of E3 Ubiquitin Ligase Cbl Proteins in Interleukin-2-Induced Jurkat T-Cell Activation

Interleukin- (IL-) 2 is the major growth factor for T-cell activation and proliferation. IL-2 has multiple functions in the regulation of immunological processes. Although most studies focus on T-cell immunomodulation, T-cell activation by IL-2 is the foundation of priming the feedback loop. Here, we investigated the effect of MAPK/ERK and PI3K/Akt signaling pathways on IL-2-induced cell activation and the regulatory mechanisms of upstream ubiquitin ligase Cbl-b and c-Cbl. Morphological analysis of Jurkat T cells was performed by cytospin preparations with Wright-Giemsa stain. CD25 expression on Jurkat T cells was determined by flow cytometry. Changes in cell activation proteins such as p-ERK, ERK, p-Akt, Akt, and ubiquitin ligase Casitas B-cell Lymphoma (Cbl) proteins were analyzed by western blot. Following IL-2-induced activation of Jurkat T cells, p-ERK expression was upregulated, while there was no change in p-Akt, ERK, or Akt expression. Thus, the MAPK/ERK signaling pathway, but not PI3K/Akt, was involved in IL-2-induced T-cell activation. Either using PD98059 (a specific inhibitor for p-ERK) or depletion of ERK with small interfering RNA (siRNA) reduced the expression of CD25. This study also showed that ubiquitin ligase proteins Cbl-b and c-Cbl might be involved in IL-2-induced Jurkat T-cell activation by negatively regulating the MAPK/ERK signaling pathway.