Introduction

(SMP) holophosphatase complex functions as a key regulator of the receptor tyrosine kinase (RTK) signaling pathway by removing an inhibitory phosphate on the RAF family of proteins to allow for MAPK signaling.^[1] This interaction of the RTK-Ras pathway and the SMP complex drives cell proliferation.^[2] 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. uses a crescent shape to enhance substrate interactions and complex stability.^[3] contains the the catalytic site of the complex which dephosphorylates the N-terminal phosphoserine (NTpS) of RAF green link here.^[3] binds to GTP which triggers assembly of the SMP complex. The C-terminus of the MRAS subunit localizes the complex to the cell membrane.^[3] WRITE ABOUT RAF HERE Mutations in one or multiple of these subunits leads to over-activation of the signaling pathway, which may result in cancer and developmental disorders called RASopathies.^[1]

There are many regulatory mechanisms that serve as a lock on this RAS-MAPK pathway, decreasing the likelihood of unintentional pathway activation. One is a protein dimer called 14-3-3 that keeps inactive RAF localized to the cytoplasm. An N-terminal phosphorylated serine (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 the 14-3-3 dimer, and activating the RAS-MAPK cell proliferation pathway.

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

Figure 2: MRAS binding sites with SHOC2, PP1C, and RAF (PDB 7DSO).^[3].

Structure of Subunits

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. ^[1]. 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

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).^[3]. 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.^[3] SHOC2 and MRAS aid in the specificity of the enzymatic activity. Hence, PP1C requires the presence of SHOC2 and MRAS to be function. ^[2] 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.^[3]. 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.^[4] ***insert what residues are mutated and HOW it leads to more activity.

PP1C activity is regulated by short linear interaction motifs or PP1C-binding regulatory proteins.^[2] The regulatory proteins bind to small linear motifs in PP1C, like RVXF.^[3] The RVXF motif and interaction site is located in PP1C through the N-terminal disordered region, which ^[1] There is a direct interaction between the RVXF motif of SHOC2 and the hydrophobic RVXF-binding pocket of PP1C.^[2][1] This hydrophobic binding site is adjacent to the catalytic metal ions. In the Ras/Raf signaling cascade, the region of Raf that is C-terminal to the phosphate group binds to this hydrophobic groove, and the remaining residues bind to the hydrophobic region of SHOC2. Raf binding to this region of SHOC2 is what allows PP1C to be specific when in the SMP complex in comparison to PP1C on its own. PP1C also has a singular cysteine (C291) present in the hydrophobic binding site in order to provide further stability to the substrate-protein interaction by forming a covalent bond to the substrate.

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

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 and 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.

The Ras-Raf signaling cascade will be inhibited without the dephosphorylation of Raf at Ser259. There is a 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 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 , allowing for the interaction of Ras and Raf, and the initiation of the signaling cascade.^[5]

Figure 2: MRAS binding sites with SHOC2, PP1C, and RAF (PDB 7DSO).^[3].

Switch I and Switch II

SHOC2-PP1C-MRAS is a central gatekeeper in receptor tyrosine kinase signaling 1. Figure 1 shows the specific pathways SHOC2-PP1C-MRAS mediates. When MRAS is bound to GDP, shown in the left of figure 1, Raf is bound to a 14-3-3 protein dimer restricting it to the cytoplasm. When MRAS-GDP is exchanged for GTP via a nucleotide exchange factor GEF, a conformational change occurs. This change figure 2, causes a shift from the open to closed conformation of Switch I. figure 3 Green link The Switch I (SWI) region is made up of residues 42-48 of the MRAS domain. 1 These residues are crucial for the binding of MRAS, SHOC2, and PP1C because MRAS undergoes a conformational change that allows for SMP complex assembly upon GTP binding. Figure 2 When GTP is bound to MRAS, it is in the “closed conformation” because hydrogen bond interactions between the γ phosphate of GTP and residues in the SWI region of MRAS cause SWI to adopt a closed conformation. The closed conformation allows for the binding of SHOC2 and PP1C because there is no steric clash GREEN LINK between the SWI region of MRAS and the surface of SHOC2 when GTP is bound. Green link. The only large-scale conformational change occurs in the MRAS subunit. When GDP is bound to the MRAS domain, it is in the “open” conformation. Green link Since the γ-phosphate is not bound to GDP, there are no hydrogen bond interactions with the oxygens of the γ-phosphate group and the MRAS SWI region, causing MRAS to adpot an "open" conformation. Since SHOC2 and PP1C do not undergo much conformational change, they are in a slow equilibrium of binding and unbinding until MRAS binds to GTP allowing MRAS to bind to SHOC2 and PP1C.

=== Cancer and Rasopathies === should we intersperse this?

Common mutations in SHOC2 and PP1C lead to amino acid changes on the interaction surfaces, that can lead to higher binding affinity.^[6] 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. Mutations in PP1C can trigger increased active site activity, increasing the RAF proteins that are active and available to bind to RAS. Universally, when this MAPK cascade is unregulated, cells are able to proliferate regardless of external signals, leading to cancer and/or RASopathies.