Ordered cell cycle progression is coordinated by cyclin dependent kinases (CDKs). CDKs often phosphorylate substrates at multiple sites clustered within disordered regions. However, for most substrates, it is not known which phosphosites are functionally important. We developed a high-throughput approach, Phosphosite Scanning, that tests the importance of each phosphosite within a multisite phosphorylated domain. We show that Phosphosite Scanning identifies multiple combinations of phosphosites that can regulate protein function and reveals specific phosphorylations that are required for phosphorylation at additional sites within a domain. We applied this approach to the yeast transcription factor Hcm1, a conserved regulator of mitotic genes that is critical for accurate chromosome segregation. Phosphosite Scanning revealed a complex CDK-regulatory circuit that mediates Cks1-dependent phosphorylation of key activating sites in vivo. These results illuminate the mechanism of Hcm1 activation by CDK and establish Phosphosite Scanning as a powerful tool for decoding multisite phosphorylated domains.

An acidic extracellular pH value (pHe) is characteristic of many cancers, in contrast to the physiologic pHe found in most benign cells. This difference in pH offers a unique opportunity to design and engineer chemicals that can be employed for pH-selective reactions in the extracellular fluid of cancer cells. The viability of human skin melanoma and corresponding fibroblasts exposed to CaS dispersions is reported. The viability of melanoma cells decreases with CaS dispersion concentration and reaches 57% at 3%, a value easily distinguishable from melanoma control experiments. In contrast, the viability of benign fibroblasts remains nearly constant within experimental error over the range of dispersion concentrations studied. The CaS dispersions facilitate vinculin delocalization in the cytoplasmic fluid, a result consistent with improved focal adhesion kinase (FAK) regulation in melanoma cells. Thermodynamic considerations are consistent with the formation of H2S from CaS in the presence of protons. The thermodynamic prediction is verified in independent experiments with solid CaS and acidic aqueous solutions. The amount of H2S formed decreases with pH. An activation energy for the process of (30 ± 10) kJ/mol in the temperature range of 280 to 330 K is estimated from initial rate measurements as a function of temperature. The total Gibbs energy minimization approach was employed to establish the distribution of sulfides—including H2S in the gas and aqueous phases—from the dissociation of CaS as a function of pH to mimic physiologically relevant pH values. Theoretical calculations suggest that partially protonated CaS in solution can be stable until the sulfur atom bonds to two hydrogen atoms, resulting in the formation of Ca2+ and H2S, which can be solvated and/or released to the gas phase. Our results are consistent with a model in which CaS is dissociated in the extracellular fluid of melanoma cells selectively. The results are discussed in the context of the potential biomedical applications of CaS dispersions in cancer therapies.

CaS/DMSO has been the subject of work in our group because it dissociates spontaneously in proton rich environments to produce Ca2+ ions and H2S. Extracellular Ca2+ concentrations in the neighbourhood of 500nM can induce apoptosis. Smaller concentrations of H2S can also induce apoptosis. We present here our efforts to establish the effect of CaS clusters in the cytoskeleton and focal adhesion points of human skin adherent benign and melanoma cells. We hypothesize that if the CaS nanoclusters can induce programmed death, then we will observe a significant impact in the focal adhesion points of cancer cells. We have studied the effect of DMSO and diluted CaS/DMSO on the focal adhesion points and cytoskeleton of cancer and benign cells using a digital confocal microscope. Anti‐vinculin monoclonal antibody and FITC‐conjugated secondary antibodies were used to detect vinculin in the cell environment. Vinculin expression was found to increase in benign cells in the first 24 and 48 hours of incubation with 1% DMSO. Comparison with control experiments the DMSO appears to localize vinculin expression around benign cell nuclei as well as the boundaries of the cytoskeleton. The CaS/DMSO increase the focal adhesion points of the adherent benign cells. In contrast, vinculin expression decreased in human melanoma fibroblasts cells incubated with 1% DMSO‐ as compared to the corresponding melanoma control cells‐ in the first 24 hours. In contrast, vinculin expression of the melanoma cells increased and de‐localized to the cell boundary edges in the first 24 hours when incubated with the CaS/DMSO. The vinculin expression in the melanoma cells incubated with 1% DMSO was found to increase significantly 48 hours as compared to the control. As with the benign cells, the melanoma cells vinculin expression was found to be more localized around the nuclei at the 48‐hour mark as compared to the first 24 hours. Vinculin expression could barely be detected in the fluorescence microscope following incubation of the melanoma cells with the CaS dispersion for 48 hours. Thus, in contrast to the benign cells, the CaS/DMSO reduce the focal adhesion points of the adherent melanoma cell. TRITC conjugated phalloidin antibody was employed to study the effect of DMSO in the F‐actin associated with the cytoskeleton of human benign and melanoma adherent skin cells. The F‐actin expression in the benign cells incubated with 1 % DMSO was found to decrease in the first 24 hours and then to increase significantly 48 hours following incubations as compared to the control. The CaS/DMSO does not seem to have any noticeable effect in the benign cells following 24 or 48 hours of incubation as compared to the control. We are led to control that the CaS/DMSO does not affect the benign skin cells cytoskeleton. The F‐actin expression changed from a protein with linear‐like to branch morphology in the melanoma cells incubated with DMSO. The CaS/DMSO increased the F‐actin branching but reduced its overall expression. In summary the results indicate that CaS does not a...

Ordered cell cycle progression is coordinated by cyclin dependent kinases (CDKs). CDKs often phosphorylate substrates at multiple sites clustered within disordered regions. However, for most substrates, it is not known which phosphosites are functionally important. We developed a high-throughput approach, Phosphosite Scanning, that tests the importance of each phosphosite within a multisite phosphorylated domain. We show that Phosphosite Scanning identifies multiple combinations of phosphosites that can regulate protein function and reveals specific phosphorylations that are required for phosphorylation at additional sites within a domain. We applied this approach to the yeast transcription factor Hcm1, a conserved regulator of mitotic genes that is critical for accurate chromosome segregation. Phosphosite Scanning revealed a complex CDK-regulatory circuit that mediates processive phosphorylation of key activating sites in vivo. These results illuminate the mechanism of Hcm1 activation by CDK and establish Phosphosite Scanning as a powerful tool for decoding multisite phosphorylated domains.

A common denominator of many cancer cells is that the extracellular pHe value is acidic (pHe<7) in contrast to the basic pHe values (>7) found in benign cells. The difference in the pHe values of cancer and benign cells has the potential to turn into a tool to develop chemical processes that will act against cancer cell proliferation selectively with a reduced impact in benign cells. We hypothesize that CaS nanostructures can selectively dissociate into Ca2+ ions and sulfides in the acidic extracellular fluid of cancer cells but not in the basic microenvironment of benign cells. We have studied the reactions of CaS as a function of pH to simulate the different extracellular environments of cancer and benign cells using the Gibbs minimization approach in the Python environment. We found that the CaS readily dissociated in the acidic pH values to Ca2+ ions and sulfides. The sulfides are distributed in pH sensitive species that include gaseous H2S, aqueous H2S, HS‐ and S2‐ ions. The results are consistent with experimental observations of H2S production from CaS as a function of pH performed in the laboratory bench. The results are discussed in the context of possible chemical process that take advantage of the acidic microenvironment in cancer tissues to selectively induce apoptosis in the cancer cells with no effect in the corresponding benign cells, reducing secondary effects associated with chemotherapies in the market today.