Caldesmon phosphorylation in actin cytoskeletal remodeling.
ABSTRACT Caldesmon is an actin-binding protein that is capable of stabilizing actin filaments against actin-severing proteins, inhibiting actomyosin ATPase activity, and inhibiting Arp2/3-mediated actin polymerization in vitro. Caldesmon is a substrate of cdc2 kinase and Erk1/2 MAPK, and phosphorylation by either of these kinases reverses the inhibitory effects of caldesmon. Cdc2-mediated caldesmon phosphorylation and the resulting dissociation of caldesmon from actin filaments are essential for M-phase progression during mitosis. Cells overexpressing the actin-binding carboxyterminal fragment of caldesmon fail to release the fragment completely from actin filaments during mitosis, resulting in a higher frequency of multinucleated cells. PKC-mediated MEK/Erk/caldesmon phosphorylation is an important signaling cascade in the regulation of smooth muscle contraction. Furthermore, PKC activation has been shown to remodel actin stress fibers into F-actin-enriched podosome columns in cultured vascular smooth muscle cells. Podosomes are cytoskeletal adhesion structures associated with the release of metalloproteases and degradation of extracellular matrix during cell invasion. Interestingly, caldesmon is one of the few actin-binding proteins that is associated with podosomes but excluded from focal adhesions. Caldesmon also inhibits the function of gelsolin and Arp2/3 complex that are essential for the formation of podosomes. Thus, caldesmon appears to be well positioned for playing a modulatory role in the formation of podosomes. Defining the roles of actin filament-stabilizing proteins such as caldesmon and tropomyosin in the formation of podosomes should provide a more complete understanding of molecular systems that regulate the remodeling of the actin cytoskeleton in cell transformation and invasion.
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ABSTRACT: Background InformationStromal cells not only play a highly important role during wound healing but also during tumour development and progression. Tumour stromal cells significantly influence the biological properties of malignant cells including their metastasising ability. The in vitro co-culture models of communication between normal fibroblasts and epithelial cells such as keratinocytes or squamous cell carcinoma cells of FaDu line representing wound healing or cancer development, were established by non-direct contact between the cells and utilised in this study to examine epithelia induced changes in overall fibroblast proteome patterns.ResultsWe were able to select the proteins co-regulated in both models in order to evaluate possible molecular commonalities between wound healing and tumour development. Amongst the most pronounced were the proteins implemented in contractile activity and formation of actin cytoskeleton such as caldesmon, calponin-2, myosin regulatory light chain 12A and cofilin-1, which were expressed independently of the presence of α-smooth muscle actin. Additionally, proteins altered differently highlighted functional and cellular phenotypes during transition of fibroblasts toward myofibroblasts or cancer-associated fibroblasts. Results showed co-ordinated regulation of cytoskeleton proteins selective for wound healing which were lost in tumorigenesis model. Vimentin bridged this group of proteins with other regulated proteins in human fibroblasts involved in protein or RNA processing and metabolic regulation.Conclusions The findings provide strong support for crucial role of stromal microenvironment in wound healing and tumorigenesis. In particular, epithelia induced protein changes in fibroblasts offer new potential targets which may lead to novel tailored cancer therapeutic strategies.This article is protected by copyright. All rights reservedBiology of the Cell 04/2014; · 3.87 Impact Factor
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ABSTRACT: In view of the great demand for human beta-cells for physiological and medical studies, we generated cell lines derived from human insulinomas which secrete insulin, C-peptide and express neuroendocrine and islet markers. In this study, we set out to characterize their proteomes, comparing them to those of primary beta-cells using DIGE followed by MS. The results were validated by Western blotting. An average of 1,800 spots was detected with less than 1% exhibiting differential abundance. Proteins more abundant in human islets, such as Caldesmon, are involved in the regulation of cell contractility, adhesion dependent signaling, and cytoskeletal organization. In contrast, almost all proteins more abundant in insulinoma cells, such as MAGE2, were first described here and could be related to cell survival and resistance to chemotherapy. Our proteomic data provides, for the first time, a molecular snapshot of the orchestrated changes in expression of proteins involved in key processes which could be correlated with the altered phenotype of human beta-cells. Collectively our observations prompt research towards the establishment of bioengineered human beta-cells providing a new and needed source of cultured human beta-cells for beta-cell research, along with the development of new therapeutic strategies for detection, characterization and treatment of insulinomas. The results obtained in the present study point at the potential value of the proteins uncovered here, since almost all proteins presenting increased levels in insulinoma cells, such as MAGE2, were first described in this study and could be related to cell survival as well as to resistance to chemotherapeutic agents. It is worth mentioning that the data obtained may also contribute to the still scarce knowledge on the molecular biology of human insulinomas. Finally, our study warrants further validation of some of these proteins in larger cohort samples as well as consideration for the development of insulinoma biomarkers and detection.Molecular and Cellular Endocrinology 07/2013; · 4.24 Impact Factor