Across the United States transition support services are lacking. The adult system in particular will require major transformation to provide the service capacity that is needed to meet the current standards of transition service accessibility for young Americans with serious mental health conditions.
Across the United States transition support services are lacking. The adult system in particular will require major transformation to provide the service capacity that is needed to meet the current standards of transition service accessibility for young Americans with serious mental health conditions.
Ras proteins are GTPases that regulate a wide range of cellular processes. The activity of Ras is dependent on its nucleotide-binding status, which is modulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Previously, we demonstrated that mutation of lysine 104 to glutamine (K104Q) attenuates the transforming capacity of oncogenic K-Ras by interrupting GEF induced nucleotide exchange. To assess the effect of this mutation in vivo, we used CRISPR/Cas9 to generate mouse models carrying the K104Q point mutation in wild-type and conditional K- RasLSL-G12D alleles. Consistent with our previous findings from in vitro studies, the oncogenic activity of K-RasG12D was significantly attenuated by mutation at K104 in vivo. These data demonstrate that lysine at position 104 is critical for the full oncogenic activity of mutant K-Ras and suggest that modification at K104, for example acetylation, may also regulate its activity. In addition, animals homozygous for K104Q were viable, fertile, and arose at Mendelian frequency, indicating that K104Q is not a complete loss of function mutation. Using biochemical and structural analysis, we found that the G12D and K104Q mutations cooperate to suppress GEF-mediated nucleotide exchange, explaining the preferential effect of K104Q on oncogenic K-Ras. Finally, we discovered an allosteric regulatory network consisting of K104 and residues including G75 on switch II (SWII) that is the key for regulating the stability of the a2 helix on SWII. In this allosteric network, K104-G75 interaction might be primary for keeping stabilization of SWII. Given the high frequency of KRAS mutations in human cancers, modulation of this network may provide a unique therapeutic approach.
Transition metal catalysts have been demonstrated in principle for temporal and spatial control of reactions, but this technology has yet to be broadly applied as a tool for biological study and cellular manipulation. Here we demonstrate the use of a ruthenium complex catalyst as a novel approach for spatial and temporal release of a pro‐drug inside cells, namely the transition of caged H89 to active H89, a Protein Kinase A (PKA) inhibitor. We synthesized caged H89 by protecting H89 as an allyl carbamate. We designed the system to intracellularly generate an active H89 inhibitor from caged H89 following addition of the ruthenium metal catalyst with the goal of inhibiting the catalytic activity of PKA and therefore reducing the phosphorylation of perilipin 1. The ruthenium catalyst activity was monitored by assessing the phosphorylation of perilipin 1, which is phosphorylated by PKA on 6 serine residues and not phosphorylated by other kinases. Experiments were performed on cell lines expressing endogenous perlipin 1, differentiated 3T3L1 cells and Y1 adrenal cells. Our results show that in its active state, H89 inhibited phosphorylation activity of PKA on substrates such as perilipin whereas the caged form of H89 lacked inhibitory activity. Experiments are planned for use of the ruthenium catalyst after the H89 and caged H89 incubation steps to determine the ability of the catalyst to activate caged H89 intracellularly. Grant Funding Source: Supported by The Royal Society of Chemistry
Ras proteins function as small GTPases to regulate a wide range of cellular processes. The activity of Ras is dependent on its nucleotide-binding status, which is modulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Previously, we demonstrated that mutation of K104 to glutamine (K104Q) attenuates the transforming capacity of ectopic oncogenic K-Ras by interrupting GEF-induced nucleotide exchange. In order to assess the biologic relevance of this mutation in vivo, we used CRISPR/Cas9 to generate mouse models carrying the K104Q point mutation in wild-type and conditional K-RasLSL-G12D alleles. Animals homozygous for K104Q were viable, fertile, and arose at Mendelian frequency, indicating that K104Q is not a complete loss-of-function mutation. Consistent with our previous findings from in vitro studies, however, the oncogenic activity of K-RasG12D was significantly attenuated by mutation at K104. These data demonstrate that lysine at position 104 is critical for the full oncogenic activity of mutant K-Ras and suggest that modification at K104, for example acetylation, may also regulate its activity. Using biochemical and structural analysis, we found that the G12D and K104Q mutations cooperate to suppress GEF-mediated nucleotide exchange, explaining the preferential effect of K104Q on oncogenic K-Ras. Finally, we discovered an allosteric regulatory network consisting of K104 and residues on switch II that is key for regulating the stability of the switch II alpha helix. Given the high frequency of KRAS mutations in human cancers, modulation of this network may provide a unique therapeutic approach. Citation Format: Moon Hee Yang, Bethany Hunt, Christian Johnson, Dhirendra Simanshu, Kevin Haigis. Mutation of K104 abrogates the oncogenic properties of K-RasG12D [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B47.
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