Sandbox Reserved 1767: Difference between revisions
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== Structure of Subunits == | == Structure of Subunits == | ||
=== SHOC2 === | === SHOC2 === | ||
The presence of SHOC2 is essential for complex formation. It a crescent shaped complex that serves as a bridge for PP1C and MRAS, maximizing interaction between the three subunits of the SMP complex. SHOC2 contains a large leucine rich region (LRR) that provides stability and localizes subunit PP1C to the membrane. Houseman SHOC2 only undergoes a 6° conformational change when PP1C and MRAS bind, showing SHOC2 is a scaffolding protein that provides a favorable interface for complex formation. SHOC2 depletion is being studied as a therapeutic approach for RAS-driven cancers due to large scale interactions of the subunits being made possible by SHOC2. <ref name="Kwon">PMID: 35831509</ref>. SHOC2 and PP1C first engage in binding with each other via an N-terminal RVXF motif on SHOC2 that is complimentary to a sequence on PP1C. SHOC2 residues V64 and F66 embed in the complimentary region of PP1C, enhancing SHOC2 affinity for PP1C. SHOC2 bind MRAS-GTP through β strands of a LRR that interacts with a hydrophobic region of MRAS-GTP further stabilizing the complex. KWON | The presence of SHOC2 is essential for complex formation. It a crescent shaped complex that serves as a bridge for PP1C and MRAS, maximizing interaction between the three subunits of the SMP complex. SHOC2 contains a large leucine rich region (LRR) that provides stability and localizes subunit PP1C to the membrane. Houseman SHOC2 only undergoes a <scene name='95/952693/Shoc2_gtp_bound_vs_gdp_bound/7'>6° conformational change</scene> when PP1C and MRAS bind, showing SHOC2 is a scaffolding protein that provides a favorable interface for complex formation. SHOC2 depletion is being studied as a therapeutic approach for RAS-driven cancers due to large scale interactions of the subunits being made possible by SHOC2. <ref name="Kwon">PMID: 35831509</ref>. SHOC2 and PP1C first engage in binding with each other via an N-terminal RVXF motif on SHOC2 that is complimentary to a sequence on PP1C. SHOC2 residues V64 and F66 '''GREEN LINK?''' embed in the complimentary region of PP1C, enhancing SHOC2 affinity for PP1C. SHOC2 bind MRAS-GTP through β strands of a LRR that interacts with a hydrophobic region of MRAS-GTP further stabilizing the complex. KWON | ||
=== PP1C === | === PP1C === | ||
The Protein phosphatase complex 1 (PP1C) subunit contains the catalytic site of the SMP complex. The PP1C subunit is a phosphatase enzyme responsible for the removal of a phosphate group on the N-terminal phosphoserine (NTpS) of RAF (Ser259).<ref name="Liau">PMID: 35768504</ref>. The exact mechanism of dephosphorylation is currently unknown, but there are three catalytic metal ions: 2 Mn2+ and 1 Cl- present that coordinate nucleophilic water molecules in the active site. This dephosphorylation event allows for pathway activation. Although PP1C can dephosphorylate other proteins independently from the SMP complex, it cannot act on Raf unless bound to the complex because it lacks intrinsic substrate selectivity.<ref name="Liau">PMID: 35768504</ref> SHOC2 and MRAS aid in the specificity of the enzymatic activity. Hence, PP1C requires the presence of SHOC2 and MRAS to be function. <ref name="Hauseman">PMID:35830882</ref> PP1C binds to SHOC2 and MRAS-GTP in a specific orientation that doesn’t change the conformation of the catalytic site and leaves it accessible for substrate binding. | The Protein phosphatase complex 1 (PP1C) subunit contains the catalytic site of the SMP complex. The PP1C subunit is a phosphatase enzyme responsible for the removal of a phosphate group on the N-terminal phosphoserine (NTpS) of RAF (Ser259).<ref name="Liau">PMID: 35768504</ref>. The exact mechanism of dephosphorylation is currently unknown, but there are three catalytic metal ions: 2 Mn2+ and 1 Cl- present that coordinate nucleophilic water molecules in the active site. This dephosphorylation event allows for pathway activation. Although PP1C can dephosphorylate other proteins independently from the SMP complex, it cannot act on Raf unless bound to the complex because it lacks intrinsic substrate selectivity.<ref name="Liau">PMID: 35768504</ref> SHOC2 and MRAS aid in the specificity of the enzymatic activity. Hence, PP1C requires the presence of SHOC2 and MRAS to be function. <ref name="Hauseman">PMID:35830882</ref> PP1C binds to SHOC2 and MRAS-GTP in a specific orientation that doesn’t change the conformation of the catalytic site and leaves it accessible for substrate binding. | ||
PP1C binds to SHOC2 through a hydrophobic n-terminal disordered region that is complimentary to the RVXF motif on SHOC2. GREEN LINK or picture? Similarly to SHOC2, PP1C does not undergo a significant conformational change when SHOC2 and MRAS-GTP bind. The lack of conformational change shows that the structure of PP1C is not dependent on the SMP complex, but in order to act as a phosphatase it must be bound to the complex.<ref name="Liau">PMID: 35768504</ref>. | PP1C binds to SHOC2 through a hydrophobic n-terminal disordered region that is complimentary to the RVXF motif on SHOC2. GREEN LINK or picture? Similarly to SHOC2, PP1C does not undergo a significant conformational change when SHOC2 and MRAS-GTP bind. The lack of conformational change shows that the structure of PP1C is not dependent on the SMP complex, but in order to act as a phosphatase it must be bound to the complex.<ref name="Liau">PMID: 35768504</ref>. | ||
PP1C binds to SHOC2 and MRAS-GTP in a specific orientation that doesn’t change the conformation of the catalytic site and leaves it accessible for substrate binding. GREEN LINK or picture? | PP1C binds to SHOC2 and MRAS-GTP in a specific orientation that doesn’t change the conformation of the catalytic site and leaves it accessible for substrate binding. '''GREEN LINK or picture?''' | ||
The substrate binds through hydrogen bonds with the main chain and side chain atoms of the catalytic residues **insert residue numbers here**. Mutations in the active site lead to increased activity, causing the Ras/Raf signaling cascade to be triggered more frequently.<ref name="Hurley">PMID: 17636256</ref> ***insert what residues are mutated and HOW it leads to more activity. | The substrate binds through hydrogen bonds with the main chain and side chain atoms of the catalytic residues **insert residue numbers here**. Mutations in the active site lead to increased activity, causing the Ras/Raf signaling cascade to be triggered more frequently.<ref name="Hurley">PMID: 17636256</ref> ***insert what residues are mutated and HOW it leads to more activity. | ||
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PP1C is involved in many different cellular signaling pathways including protein synthesis, muscle contraction, and even carbohydrate metabolism. Wolfgang In all these pathways, including the SMP pathway, PP1C does not exist as a monomer, it is present in holoenzyme form complex with one of two regulatory subunits ensuring there is no sporadic pathway activation. Schulman | PP1C is involved in many different cellular signaling pathways including protein synthesis, muscle contraction, and even carbohydrate metabolism. Wolfgang In all these pathways, including the SMP pathway, PP1C does not exist as a monomer, it is present in holoenzyme form complex with one of two regulatory subunits ensuring there is no sporadic pathway activation. Schulman | ||
===RAS/RAF === | ===RAS/RAF === | ||
=RAF= | |||
While RAF is not technically part of the SMP protein complex, it is crucial for advancement in the cell signaling pathway SMP helps mediate. RAF plays many different roles in this pathway and has many different domains. RAF has a RAS binding domain (RBD), a N-terminal phosphorylated serine (NTpS), and a kinase domain. Figure ?? shows these domains and mechanistically how RAF is involved in signal advancement | [[Image:pic3.jpg|250 px|right|thumb|Figure 2: MRAS binding sites with SHOC2, PP1C, and RAF (PDB 7DSO).<ref name="Liau">PMID: 35768504</ref>.]] | ||
==RAF== | |||
While RAF is not technically part of the SMP protein complex, it is crucial for advancement in the cell signaling pathway SMP helps mediate. RAF plays many different roles in this pathway and has many different domains. RAF has a RAS binding domain (RBD), a N-terminal phosphorylated serine (NTpS), and a kinase domain. Figure ?? shows these domains and mechanistically how RAF is involved in signal advancement or lack thereof. When its N-terminal serine is phosphorylated RAF is bound to a 14-3-3 protein dimer, inactivating the pathway. Whenever the SMP complex is assembled, PP1C dephosphorylates this serine starting the signaling cascade. | |||
==RAS== | |||
The Ras-Raf signaling cascade will be inhibited without the dephosphorylation of Raf at Ser259. 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 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/7'>autoinhibited confirmation</scene> as 14-3-3 restricts Raf to the cytoplasm and sterically inhibits Raf from binding with activated Ras. This interaction is crucial in regulating cell proliferation, as it prevents cell growth in the absence of a signal. Extracellular growth factors trigger GTP to bind to MRAS, which triggers SMP formation. 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. Once dephosphorylated, Raf is in the <scene name='95/952695/Non-inhibited_confirmation/9'>active confirmation</scene>, allowing for the interaction of Ras and Raf, and the initiation of the signaling cascade.<ref name="Young">PMID: 30348783</ref> | The Ras-Raf signaling cascade will be inhibited without the dephosphorylation of Raf at Ser259. 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 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/7'>autoinhibited confirmation</scene> as 14-3-3 restricts Raf to the cytoplasm and sterically inhibits Raf from binding with activated Ras. This interaction is crucial in regulating cell proliferation, as it prevents cell growth in the absence of a signal. Extracellular growth factors trigger GTP to bind to MRAS, which triggers SMP formation. 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. Once dephosphorylated, Raf is in the <scene name='95/952695/Non-inhibited_confirmation/9'>active confirmation</scene>, allowing for the interaction of Ras and Raf, and the initiation of the signaling cascade.<ref name="Young">PMID: 30348783</ref> | ||
