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[[Image:MECH.png|700 px|center|thumb|'''Figure 1''': Mechanism of SMP complex formation and activation of RAF.<ref name="Liau">PMID: 35768504</ref><ref name="Lavoie">PMID: 35970881</ref>]] | [[Image:MECH.png|700 px|center|thumb|'''Figure 1''': Mechanism of SMP complex formation and activation of RAF.<ref name="Liau">PMID: 35768504</ref><ref name="Lavoie">PMID: 35970881</ref>]] | ||
The RAS-RAF signaling cascade is inhibited when RAF is phosphorylated at Ser259.<ref name="Kwon">PMID: 35831509</ref> There is a <scene name='95/952695/14-3-3/1'>14-3-3</scene> dimer present in the cytoplasm that interacts with RAF through [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4005716/. hydrogen bonds] between R129 of 14-3-3 and Ser259 of RAF when Ser259 is phosphorylated. This interaction causes an <scene name='95/952695/Autoinhibited_confirmation/9'>autoinhibited confirmation</scene> as 14-3-3 restricts RAF to the cytoplasm and sterically inhibits RAF from binding with RAS. This interaction is crucial in regulating [https://us.progen.com//Antibodies/Research-Area/Cell-Cycle-Proliferation/ cell proliferation], as it prevents cell growth in the absence of a signal. [https://www.ncbi.nlm.nih.gov/books/NBK26877/#:~:text=Extracellular%20Growth%20Factors%20Stimulate%20Cell,Cell%20Growth%2C%20Cell%20Division%2C%20and Extracellular Growth Factors] cause GTP to bind to MRAS which triggers SMP formation <ref name="Lavoie">PMID: 35970881</ref>.Upon SMP complex formation, PP1C is brought into close proximity of RAS, leading to the dephosphorylation of Ser259 of RAF by the active site of PP1C <ref name="Lavoie">PMID: 35970881</ref>. Once dephosphorylated, RAF is in the | The RAS-RAF signaling cascade is inhibited when RAF is phosphorylated at Ser259.<ref name="Kwon">PMID: 35831509</ref> There is a <scene name='95/952695/14-3-3/1'>14-3-3</scene> dimer present in the cytoplasm that interacts with RAF through [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4005716/. hydrogen bonds] between R129 of 14-3-3 and Ser259 of RAF when Ser259 is phosphorylated. This interaction causes an <scene name='95/952695/Autoinhibited_confirmation/9'>autoinhibited confirmation</scene> as 14-3-3 restricts RAF to the cytoplasm and sterically inhibits RAF from binding with RAS. This interaction is crucial in regulating [https://us.progen.com//Antibodies/Research-Area/Cell-Cycle-Proliferation/ cell proliferation], as it prevents cell growth in the absence of a signal. [https://www.ncbi.nlm.nih.gov/books/NBK26877/#:~:text=Extracellular%20Growth%20Factors%20Stimulate%20Cell,Cell%20Growth%2C%20Cell%20Division%2C%20and Extracellular Growth Factors] cause GTP to bind to MRAS which triggers SMP formation <ref name="Lavoie">PMID: 35970881</ref>.Upon SMP complex formation, PP1C is brought into close proximity of RAS, leading to the dephosphorylation of Ser259 of RAF by the active site of PP1C <ref name="Lavoie">PMID: 35970881</ref>. Once dephosphorylated, RAF is in the | ||
<scene name='95/952695/Non-inhibited_confirmation/ | <scene name='95/952695/Non-inhibited_confirmation/11'>active confirmation</scene>, allowing RAS to bind RAF, initiating the signaling cascade.<ref name="Young">PMID: 30348783</ref> | ||
== Structure of Subunits == | == Structure of Subunits == | ||
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==RAS== | ==RAS== | ||
RAS proteins are GTP-dependent [https://pubmed.ncbi.nlm.nih.gov/14604583/. intracellular switches] that are anchored to the plasma membrane. <ref name="Liau">PMID: 35768504</ref> RAS proteins activate RAF kinases through direct binding and membrane recruitment, resulting in RAF dimerization and pathway activation <ref name="Liau">PMID: 35768504</ref>. The SMP complex has specificity for MRAS. Other RAS proteins may bind to SHOC2, but MRAS induces the complex formation with a significantly lower [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004624/. dissociation constant] <ref name="Liau">PMID: 35768504</ref>. There are no known membrane interacting regions on SHOC2 and PP1C, meaning the [https://bio.libretexts.org/Bookshelves/Introductory_and_General_Biology/Book%3A_General_Biology_(Boundless)/03%3A_Biological_Macromolecules/3.05%3A_Lipid_Molecules_-_Phospholipids#:~:text=The%20fatty%20acid%20chains%20are,the%20intracellular%20and%20extracellular%20fluid. hydrophobic fatty acid tail] on MRAS is responsible for recruiting the complex to the cell membrane | RAS proteins are GTP-dependent [https://pubmed.ncbi.nlm.nih.gov/14604583/. intracellular switches] that are anchored to the plasma membrane. <ref name="Liau">PMID: 35768504</ref> RAS proteins activate RAF kinases through direct binding and membrane recruitment, resulting in RAF dimerization and pathway activation <ref name="Liau">PMID: 35768504</ref>. The SMP complex has specificity for MRAS. Other RAS proteins may bind to SHOC2, but MRAS induces the complex formation with a significantly lower [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004624/. dissociation constant] <ref name="Liau">PMID: 35768504</ref>. There are no known membrane interacting regions on SHOC2 and PP1C, meaning the [https://bio.libretexts.org/Bookshelves/Introductory_and_General_Biology/Book%3A_General_Biology_(Boundless)/03%3A_Biological_Macromolecules/3.05%3A_Lipid_Molecules_-_Phospholipids#:~:text=The%20fatty%20acid%20chains%20are,the%20intracellular%20and%20extracellular%20fluid. hydrophobic fatty acid tail] on MRAS is responsible for recruiting the complex to the cell membrane. This allows only for 2D movement and increasing local concentrations of the players needed in this signaling pathway <ref name="Hauseman">PMID:35830882</ref>. | ||
A significant amount of steric overlap is seen in MRAS for the binding sites of PP1C, SHOC2, and RAF <ref name="Liau">PMID: 35768504</ref>. In '''Figure 3''', {{Font color|lime|MRAS}} is shown in green, with the {{Font color|cyan|SHOC2 binding site}} colored cyan, the {{Font color|violet|PP1C binding site}} colored violet, and the {{Font color|red|RAF binding site}} shown in red on a different RAS protein. Hence, multiple RAS proteins are required for further activation of the receptor tyrosine kinase pathway <ref name="Lavoie">PMID: 35970881</ref>. Due to the significant overlap in binding domains, one MRAS molecule is needed to recruit SHOC2 and PP1C to the membrane, and another RAS molecule is needed activate RAF <ref name="Lavoie">PMID: 35970881</ref>. The ability of MRAS-GTP to cluster at the cell membrane is a crucial capability for this protein complex. The presence of this <scene name='95/952695/413cellmemprotrusion/4'>palmitoylated tail</scene> is responsible for this anchoring to the cell membrane, similar to the hydrophobic fatty acid tail on MRAS that is responsible for recruiting SMP to the cell membrane. | A significant amount of steric overlap is seen in MRAS for the binding sites of PP1C, SHOC2, and RAF <ref name="Liau">PMID: 35768504</ref>. In '''Figure 3''', {{Font color|lime|MRAS}} is shown in green, with the {{Font color|cyan|SHOC2 binding site}} colored cyan, the {{Font color|violet|PP1C binding site}} colored violet, and the {{Font color|red|RAF binding site}} shown in red on a different RAS protein. Hence, multiple RAS proteins are required for further activation of the receptor tyrosine kinase pathway <ref name="Lavoie">PMID: 35970881</ref>. Due to the significant overlap in binding domains, one MRAS molecule is needed to recruit SHOC2 and PP1C to the membrane, and another RAS molecule is needed activate RAF <ref name="Lavoie">PMID: 35970881</ref>. The ability of MRAS-GTP to cluster at the cell membrane is a crucial capability for this protein complex. The presence of this <scene name='95/952695/413cellmemprotrusion/4'>palmitoylated tail</scene> is responsible for this anchoring to the cell membrane, similar to the hydrophobic fatty acid tail on MRAS that is responsible for recruiting SMP to the cell membrane. | ||
MRAS contains two regions called Switch I (SWI) and Switch II (SWII) that undergo conformational changes depending if MRAS is bound to GDP or GTP <ref name="Liau">PMID: 35768504</ref>. The conformation of these switches determines if the SMP complex can form or not. Mutations to MRAS can lead to consistent GTP-loading, causing an increase in the formation of the SMP complex | MRAS contains two regions called Switch I (SWI) and Switch II (SWII) that undergo conformational changes depending if MRAS is bound to GDP or GTP <ref name="Liau">PMID: 35768504</ref>. The conformation of these switches determines if the SMP complex can form or not. Mutations to MRAS can lead to consistent GTP-loading, causing an increase in the formation of the SMP complex as well as consistent activation of the cell-proliferation pathway in the absence of external growth factors. | ||
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=== Cancer and Rasopathies === | === Cancer and Rasopathies === | ||
Common mutations in SHOC2 and PP1C lead to amino acid changes on the interaction surfaces, | Common mutations in SHOC2 and PP1C lead to amino acid changes on the interaction surfaces, which can result in [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2692211/. higher binding affinity].<ref name="Lavoie">PMID: 35970881</ref>The interface of SHOC2-PP1C is stabilized by the <scene name='95/952695/Q249k_mutation/1'>Q249K</scene> mutation because this creates a salt bridge with E116 of PP1C. This enhances the binding energy by -22.7 kcal/mol. Mutations to MRAS can result in consistent GTP-loading, increasing the formation of the SMP complex in the absence of external growth factors that are necessary for activation of the pathway in a healthy organism. The majority of wild type MRAS in cells are bound to GDP, whereas the MRAS with the Q71L mutation locked MRAS in the GTP bound state.<ref name="Hauseman">PMID:35830882</ref> In MRAS, <scene name='95/952695/Q249k_mutation/2'>Q71L and G23V</scene> both show increased interaction with other effectors such as BRAF, CRAF, and AF6, consistent with gain-of-function mutations that activate MRAS, leading to GTP-loading. | ||
Mutations in PP1C can trigger increased active site activity, increasing the RAF proteins that are active and available to bind to RAS. In patients with [https://medlineplus.gov/genetics/condition/noonan-syndrome/#:~:text=Noonan%20syndrome%20is%20a%20condition,many%20other%20signs%20and%20symptoms. Noonan Syndrome], a disease in the RASopathy family, a point mutation of <scene name='95/952695/Q249k_mutation/2'>T68I</scene> MRAS was identified, however | Mutations in PP1C can trigger increased active site activity, increasing the RAF proteins that are active and available to bind to RAS. In patients with [https://medlineplus.gov/genetics/condition/noonan-syndrome/#:~:text=Noonan%20syndrome%20is%20a%20condition,many%20other%20signs%20and%20symptoms. Noonan Syndrome], a disease in the RASopathy family, a point mutation of <scene name='95/952695/Q249k_mutation/2'>T68I</scene> MRAS was identified, however the effects this has are unknown.<ref name="Young">PMID: 30348783</ref> Universally, when this MAPK cascade is unregulated, cells are able to proliferate regardless of external signals, leading to [https://www.ncbi.nlm.nih.gov/books/NBK20362/. cancer] and/or RASopathies. | ||