Sandbox Reserved 1767: Difference between revisions

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<scene name='95/952695/Overall_image/2'>The SHOC2-MRAS-PP1C</scene> (SMP) holophosphatase complex functions as a key regulator of the [https://www.nature.com/scitable/topicpage/rtk-14050230/#:~:text=One%20of%20the%20most%20common,anchored%20to%20the%20plasma%20membrane. receptor tyrosine kinase (RTK)] signaling pathway by removing an inhibitory phosphate on the [https://www.sciencedirect.com/science/article/pii/S0167488907001164. RAF] family of proteins to allow for [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536342/. MAPK signaling].<ref name="Kwon">PMID: 35831509</ref> This interaction of the RTK-RAS pathway and the SMP complex drives cell proliferation.<ref name="Hauseman">PMID:35830882</ref> The SMP complex is made of three subunits, SHOC2, PP1C, and MRAS. Each of these subunits has a different shape that corresponds to its different function. <scene name='95/952695/Shoc2intro/1'>The SHOC2 subunit</scene> uses a crescent shape to enhance substrate interactions and complex stability.<ref name="Liau">PMID: 35768504</ref> <scene name='95/952695/Pp1cintro/3'>The PP1C subunit</scene> contains the the catalytic site of the complex which dephosphorylates the N-terminal phosphoserine (NTpS) of RAF.<ref name="Liau">PMID: 35768504</ref> <scene name='95/952694/Pp1ccorrectintro/1'>The MRAS subunit</scene> binds to GTP which causes assembly of the SMP complex. The <scene name='95/952695/413cellmemprotrusion/4'>C-terminus of MRAS</scene> localizes the complex to the cell membrane.<ref name="Liau">PMID: 35768504</ref> Once the SMP compelx is assembled, MRAS can bind to <scene name='95/952695/Raf/3'>RAF</scene>, allowing the [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5000522/. signaling cascade] to continue. Mutations in one or multiple of these subunits can lead to over-activation of the signaling pathway, which may result in cancer and developmental disorders called [https://kidshealth.org/en/parents/rasopathies.html RASopathies].<ref name="Kwon">PMID: 35831509</ref>

There are many regulatory mechanisms that serve as a lock on this [https://www.cancer.gov/research/key-initiatives/ras/about#:~:text=RAS%20proteins%20are%20important%20for,inactive%20(GDP%20form)%20states. RAS]-[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536342/. MAPK] pathway, decreasing the likelihood of unintentional pathway activation. <ref name="Hauseman">PMID:35830882</ref> One example is <scene name='95/952695/14-3-3/1'>14-3-3</scene>, a protein dimer that keeps inactive RAF localized to the cytoplasm. An <scene name='95/952695/Raf_ntps/1'>N-terminal phosphorylated serine</scene> (NTpS) keeps RAF bound to this protein dimer, and when the SMP complex is assembled, the catalytic subunit, PP1C, removes the phosphate group from ~~the serine residue~~, releasing RAF from <scene name='95/952695/14-3-3/1'>14-3-3</scene>, and activating the RAS-MAPK cell proliferation pathway. <ref name="Hauseman">PMID:35830882</ref>

There are many regulatory mechanisms that serve as a lock on this [https://www.cancer.gov/research/key-initiatives/ras/about#:~:text=RAS%20proteins%20are%20important%20for,inactive%20(GDP%20form)%20states. RAS]-[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536342/. MAPK] pathway, decreasing the likelihood of unintentional pathway activation. <ref name="Hauseman">PMID:35830882</ref> One example is <scene name='95/952695/14-3-3/1'>14-3-3</scene>, a protein dimer that keeps inactive RAF localized to the cytoplasm. An <scene name='95/952695/Raf_ntps/1'>N-terminal phosphorylated serine</scene> (NTpS) keeps RAF bound to this protein dimer, and when the SMP complex is assembled, the catalytic subunit, PP1C, removes the phosphate group from Ser259, releasing RAF from <scene name='95/952695/14-3-3/1'>14-3-3</scene>, and activating the RAS-MAPK cell proliferation pathway. <ref name="Hauseman">PMID:35830882</ref>

In all images and animations, {{Font color|cyan|SHOC2}} will be shown as cyan blue, {{Font color|lime|MRAS}} as lime, and {{Font color|violet|PP1C}} as violet. Other important components involved in the function of the SMP complex include the {{Font color|salmon|14-3-3}} dimer and {{Font color|slateblue|Raf}}, which will be shown in salmon and slate-blue, respectively.

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===SMP Complex Mechanism===

[[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/8'>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/~~Autoinhibited_confirmation~~/8'>active confirmation</scene>, allowing RAS to bind RAF, initiating the signaling cascade.<ref name="Young">PMID: 30348783</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

<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 ==

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==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 ~~allowing~~ only for 2D movement and increasing local concentrations of the players needed in this signaling pathway <ref name="Hauseman">PMID:35830882</ref>.

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.

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 ~~and there is~~ consistent activation of the cell-proliferation pathway in the absence of external growth factors.

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 ===

Common mutations in SHOC2 and PP1C lead to amino acid changes on the interaction surfaces, ~~that~~ can ~~lead to~~ [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. ~~The G63R mutation on SHOC2 creates two additional hydrogen bonds with D242 on PP1C, releasing another 18.88kcal/mol of interaction energy.<ref name="Kwon">PMID: 35831509</ref>~~ 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 ~~were in the~~ GDP ~~state~~, whereas the MRAS with the Q71L mutation locked in GTP~~-induced RAS conformational changes~~.<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.

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, the ~~outcome is~~ 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.

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.

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@@ Line 7: / Line 7: @@
 <scene name='95/952695/Overall_image/2'>The SHOC2-MRAS-PP1C</scene> (SMP) holophosphatase complex functions as a key regulator of the [https://www.nature.com/scitable/topicpage/rtk-14050230/#:~:text=One%20of%20the%20most%20common,anchored%20to%20the%20plasma%20membrane. receptor tyrosine kinase (RTK)] signaling pathway by removing an inhibitory phosphate on the [https://www.sciencedirect.com/science/article/pii/S0167488907001164. RAF] family of proteins to allow for [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536342/. MAPK signaling].<ref name="Kwon">PMID: 35831509</ref> This interaction of the RTK-RAS pathway and the SMP complex drives cell proliferation.<ref name="Hauseman">PMID:35830882</ref> The SMP complex is made of three subunits, SHOC2, PP1C, and MRAS. Each of these subunits has a different shape that corresponds to its different function. <scene name='95/952695/Shoc2intro/1'>The SHOC2 subunit</scene> uses a crescent shape to enhance substrate interactions and complex stability.<ref name="Liau">PMID: 35768504</ref> <scene name='95/952695/Pp1cintro/3'>The PP1C subunit</scene> contains the the catalytic site of the complex which dephosphorylates the N-terminal phosphoserine (NTpS) of RAF.<ref name="Liau">PMID: 35768504</ref> <scene name='95/952694/Pp1ccorrectintro/1'>The MRAS subunit</scene> binds to GTP which causes assembly of the SMP complex. The <scene name='95/952695/413cellmemprotrusion/4'>C-terminus of MRAS</scene> localizes the complex to the cell membrane.<ref name="Liau">PMID: 35768504</ref> Once the SMP compelx is assembled, MRAS can bind to <scene name='95/952695/Raf/3'>RAF</scene>, allowing the [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5000522/. signaling cascade] to continue. Mutations in one or multiple of these subunits can lead to over-activation of the signaling pathway, which may result in cancer and developmental disorders called [https://kidshealth.org/en/parents/rasopathies.html RASopathies].<ref name="Kwon">PMID: 35831509</ref>
-There are many regulatory mechanisms that serve as a lock on this [https://www.cancer.gov/research/key-initiatives/ras/about#:~:text=RAS%20proteins%20are%20important%20for,inactive%20(GDP%20form)%20states. RAS]-[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536342/. MAPK] pathway, decreasing the likelihood of unintentional pathway activation. <ref name="Hauseman">PMID:35830882</ref> One example is <scene name='95/952695/14-3-3/1'>14-3-3</scene>, a protein dimer that keeps inactive RAF localized to the cytoplasm. An <scene name='95/952695/Raf_ntps/1'>N-terminal phosphorylated serine</scene> (NTpS) keeps RAF bound to this protein dimer, and when the SMP complex is assembled, the catalytic subunit, PP1C, removes the phosphate group from the serine residue, releasing RAF from <scene name='95/952695/14-3-3/1'>14-3-3</scene>, and activating the RAS-MAPK cell proliferation pathway. <ref name="Hauseman">PMID:35830882</ref>
+There are many regulatory mechanisms that serve as a lock on this [https://www.cancer.gov/research/key-initiatives/ras/about#:~:text=RAS%20proteins%20are%20important%20for,inactive%20(GDP%20form)%20states. RAS]-[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536342/. MAPK] pathway, decreasing the likelihood of unintentional pathway activation. <ref name="Hauseman">PMID:35830882</ref> One example is <scene name='95/952695/14-3-3/1'>14-3-3</scene>, a protein dimer that keeps inactive RAF localized to the cytoplasm. An <scene name='95/952695/Raf_ntps/1'>N-terminal phosphorylated serine</scene> (NTpS) keeps RAF bound to this protein dimer, and when the SMP complex is assembled, the catalytic subunit, PP1C, removes the phosphate group from Ser259, releasing RAF from <scene name='95/952695/14-3-3/1'>14-3-3</scene>, and activating the RAS-MAPK cell proliferation pathway. <ref name="Hauseman">PMID:35830882</ref>
 In all images and animations, {{Font color|cyan|SHOC2}} will be shown as cyan blue, {{Font color|lime|MRAS}} as lime, and {{Font color|violet|PP1C}} as violet. Other important components involved in the function of the SMP complex include the {{Font color|salmon|14-3-3}} dimer and {{Font color|slateblue|Raf}}, which will be shown in salmon and slate-blue, respectively.
@@ Line 13: / Line 13: @@
 ===SMP Complex Mechanism===
 [[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/8'>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/Autoinhibited_confirmation/8'>active confirmation</scene>, allowing RAS to bind RAF, initiating the signaling cascade.<ref name="Young">PMID: 30348783</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
+<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 ==
@@ Line 32: / Line 33: @@
 ==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 allowing only for 2D movement and increasing local concentrations of the players needed in this signaling pathway <ref name="Hauseman">PMID:35830882</ref>.
+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.
-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 and there is consistent activation of the cell-proliferation pathway in the absence of external growth factors.
+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 ===
-Common mutations in SHOC2 and PP1C lead to amino acid changes on the interaction surfaces, that can lead to [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. The G63R mutation on SHOC2 creates two additional hydrogen bonds with D242 on PP1C, releasing another 18.88kcal/mol of interaction energy.<ref name="Kwon">PMID: 35831509</ref> 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 were in the GDP state, whereas the MRAS with the Q71L mutation locked in GTP-induced RAS conformational changes.<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.
+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, the outcome is 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.
+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.