Protein structures and catalytic mechanisms. The Phosphoinositide 3-kinases

Protein kinases are a large family of enzymes that catalyze the transfer of phosphate from ATP to serine, threonine, and tyrosine residues of their substrate proteins. Protein kinases are found in all eukaryotes from yeast to mammals. They are involved in many aspect of cell as they play a critical role in signaling and other major cellular processes. While each specific kinase is thought to have a specific function, there are many conserved domains among kinases regarding their structures and catalytic mechanisms. The Phosphoinositide 3-kinases (PI3Ks) related protein kinases (PIKKs) are a family of protein kinases with a large range of important cellular functions. PI3Ks phosphorylate the inositol ring on the 3 position, which creates a docking site for proteins. There are eight catalytic PI3K subunits that are divided into three classes based on the sequence alignment and domain structures (Fig.1: a,b). The class III PI3K is the oldest PI3K and is the only one found in yeast and plants with a Vps34 domain structure that phosphorylates phosphatidylinositol to generate PI3P. The class II PI3Ks with a CII  domain structure are localized in endosomes, but their function is still not well understood. The class I PI3Ks are heterodimeric proteins with a p110 domain structure. The class I PI3K subunits are further subdivided into class IA and IB. the class IA subunits are associated with a SH2-containing regulatory subunits of PIP3. PIP3 acts as a membrane tether for a subset of proteins with one or more pleckstrin homology (PH) domains. PH domains need to have enough affinity for the PIP3 in order for it to be selectively regulated by class I PI3Ks. In mammals, about 40 of the 200+ proteins with PH domains can be controlled by PIP3. However most of the focus is contributed to the regulation of the Akt pathway and its role in controlling the activation of the mammalian target of rapamycin (mTOR). mTOR is  a serine/threonine kinase in the PI3K-related kinase (PIKK). It is a central regulator of cellular metabolism, growth and survival in response to hormones, growth factors, nutrients, energy, and stress signals. mTOR directly or indirectly regulates the phosphorylation of many proteins. It functions as part of two structurally and functionally distinct signaling complexes mTOR complex 1 (mTORC1) and mTOR complex2 (mTORC2). Activated mTORC1 upregulates protein synthesis and is mainly involved in cell growth (Fig. 2.a). It is defined by its three core components: mTOR, Raptor (regulator associated with mTOR), and mLST8.  Raptor facilitates substrate recruitment to mTORC1 while the mLST8 associate with the catalytic domain of mTORC1, which stabilize the kinase activation loop that is essential for mTORC1 function (figure2b). mTORC2 may regulate other cellular processes including the organization of the cytoskeleton. It plays a critical role in the phosphorylation of AKT1, a pro-survival effector of PI3K (Fig. 2.a). mTOR2 also contains  mTOR and mSLT8, but instead of Raptor mTORC2 contains Rictor, a rapamycin insensitive companion of mTOR (Fig. 2.c).

·      Overall structure of mTOR kinase.

The mTOR protein is a 289-KDa that belongs to the PI3K-related kinase family and is conserved throughout evolution with a kinase domain similar to the PI3Ks. The conserved N-terminal of the mTOR kinase domain, long helical repeats, is shared among all PIKKs. Recently, a crystal structure of the mTOR kinase domain in complex with mST8 has been resolved at 3.2  resolution (Fig.3). The structure shows the two-lobe catalytic core found in both mTORC1 and mTORC2. It also shows the FRB (FK506-rapamycin binding) domain, the FATC (FRAPP, ATM, TOR at C-terminus) domain, the LBE (LST8 binding element), and the KAL (activation loop helix) are all PIKK specific features. Also, the crystal structure (Fig.3) shows a potion of he N-teminal helical repeats with the FAT domain. The interaction between the Kinase domain (KD) and the FAT domain is established through hydrogen bonds which is thought to be important for the kinase domain structure and activity, and are common features of all PIKKs.

There are three distinct clusters of activating mutation located within the kinase domain and in the interface between the kinase domain and the FAT domain. Mutations in this area are believed to make the end of the catalytic cleft less protected by either weakening the interaction between the helices or decreasing the kinase domain interactions. This allows mTOR to become more active toward the physiological substrates 4E-BP1 and S6K1 as well as increases its kinase activity by having more access to the catalytic site.

One side of the activation loop packs with the k9b insertion, and the other side packs with FATC (Fig.2b). The FATC’s N-terminal half forms a helix (kthat is present in the PI3K structures, but its C-terminal half is absent from the PI3Ks. The FATC’s C-terminal forms three short helices that pack with the activation loop on one side and with the LBE on the other side (Fig.2b). The FATC and activation loop sequences are conserved among the PIKKs, but not the LBE. However, all PIKK family members contain an LBE-like insertion that may similarly pack with FATC.

·      Overview of mTOR signaling pathway.

mTOR interacts with many proteins to form at least the two distinct multiprotein mTORC1 and mTORC2 . The mTOR complexes have differences in their sensitivities to rapamycin, in the upstream signals they integrate, in the substrates they regulate, and in the biological process they control. mTORC1 activity is controlled by the small GTPase Rheb. The GTPase-activating domain of Tuberin (Tsc2) increases the rate of hydrolysis of Rheb-bound GTP, rendering Rheb to the inactive GDP-bound form. Tsc2 is inhibited when it is phosphorylated, which allow Akt to release Rheb from the inhibition by Tsc2 and allows GTP-Rheb to activate mTORC1 (Fig. 4a). When there is sufficient amino acid and ATP available in the cell, mTOR is activated. However, when AMPK (plays a role in cellular homeostasis) is active it inhibits mTOR (Fig.4a). The activation of mTOR involves the assembly of proteins of the Rag GTPase family at the lysosome. The best-characterized substrates for mTORC1 are ribosomal S6 kinase (S6K) and the initiation factor 4E binding protein1 (4E-BP1). The phosphorylated form of S6K and 4E-BP1 promotes protein synthesis. S6K1phosphorylates and activates several substrates that promote mRNA translation initiation (Fig. 4b). mTORC1 also facilitates growth by promoting a shift in glucose metabolism from oxidative phosphorylation to glycolysis, which facilitates the incorporation of nutrients into new biomass (Fig.4b). Furthermore, mTORC1 leads to increased flux through the oxidative pentose phosphate pathway (PPP), which use carbons from glucose to generate the NADPH and other intermediary metabolites needed for proliferation and cell growth. In addition, mTORC1 also promotes growth by suppressing protein catabolism (Fig.2a), precisely autophagy. When the cell is under starving conditions, mTORC1 phosphorylates ULK1, a kinase that drives autophagosome formation, which prevents its activation by AMPK (Fig4.b). mTORC1 can also negatively regulate class I PI3K signaling via different mechanisms, including phosphorylation of receptors.

While mTORC1 regulates cell growth and metabolism, mTORC2 instead controls proliferation and survival primarily by phosphorylating several members of the AGC (PKA/PKG/PKC) family protein kinases (Fig. 4c). Recently, it has been shown that mTORC2 can also phosphorylate different types of PKCs, which regulates different aspects of cytoskeletal remodeling and cell migration. Moreover, mTORC2 most important role is to phosphorylate and activate Akt. Activated Akt plays important roles in the cell such as cell survival, proliferation, and growth through the phosphorylation and inhibition of different substrates like Foxo and the mTORC1 inhibitor TSC2 (Fig. 1a). Even-though the signaling pathways that lead to mTORC2 activation are not well characterized, it is considered that mTORC2 kinase activity and AKT phosphorylation at Ser473 (Fig.4a) increases activity due to the growth factors. With the growth factors simulation, AKT is phosphorylated at the cell membrane through the binding of ptdINS to its pleckstrin homology (PH) domain. Under these conditions, PDK1 is also recruited to the membrane through its PH domain and phosphorylate AKT at Ser308 (Fig. 4a).

 II.         Structure and molecular mechanism 

 

·      The  , and FAT domain structure.

The  is a human mTOR with a truncated N-terminal that is bound mLST8. The kinase activity of  is comparable to that of mTORC1, however the complex is more active at higher substrate concentrations, which is opposite to the mTORC1. The  has a compact shape with a FAT domain, which consist of an alpha-alpha helical repeats that forms a C-shaped solenoid which wraps half way around the kinase domain and clamps into it. The FATC domain is integral to the kinase domain structure. However, the mLST8 and the FRB domain extend beyond the kinase domain, thus are found on the opposite sides of the catalytic cleft (Fig. 5a). The mTOR kinase domain consists of the two-lobe structure, the N-terminal lobe (N-lobe) and the C-terminal lobe (C-lobe), which are features of both PI3K and PK families. Between the N-lobe and the C-lobe there is cleft that binds to ATP. The FRB domain, with the residue insertion, is inserted within the kinase N-lobe as well as a 40-residue insertion in the C-lobe that forms the binding site for mLST8 (Fig.5a).

The mTOR kinase domain structure starts before the FRB domain with the long k1 helix integrated to the structure of the N-lobe. The FRB insertion occurs right after the k1 helix followed by a short beta-strands and two short helices that pack the FRB. The C-lobe contains the catalytic cleft with four structural insertions (LBE, k9b, and FATC; Fig. 3b). These will form a center of interaction ate the activation loop that is important for the regulation of PK. The activation loop, which is well ordered on mTOR structure, is believed to have a comparable role in PI3Ks among which it is ordered in only the class III PIK3C3 structures.  The FATC makes interactions with the activation loop suggesting that it may have a role in stabilizing the activation loop structure and the LBE (Fig. 3b). mLST8 consists of  a seven Beta-propeller that extends the WD40 repeats and binds to both helices and the intervening loop of the LBE. mLST8 is thought to be a requisite activating subunit of mTOR complex where its surface may directly stabilize the LBE structure and indirectly influence the organization of the active site through the LBE/FATC/activation-loop spine of interaction. The FAT domain contain 28 alpha helices where alpha1 –alpha22 belong to the TRP repeat family and form three discontinuous domains (TRD1, TRD2, and TRD3). The contacts of TRD1 and HDR to the KD are important for the function and structure of mTOR (Fig. 5b).  The TRD1 and HDR segments correspond to the FAT segments, which are conserved within the PIKK family members. The FAT domain clamping onto the KD is a common feature of this family.

·      Comparison of the mTOR catalytic center to PI3Ks.

In the PI3Ks and other Pks, the N-lobe of the mTOR kinase domain is smaller. It id composed of five Beta-sheets associate with few alpha-helices while the C-lobe mostly contain alpha-helices. The active site and the ATP-binding region of the mTOR are located between the N- lobe and the C-lobe. In mTOR, the kinase domain N-lobe is structurally more similar to PI3Ks than Pks. In PI3Ks and mTOR the N-lobe packs against the HEAT repeats of the helical FAT domain.

Among kinase families, there is a conserved element of the active site known as the P-loop that is found close to the N-terminus of the conserved N-lobe. Residues from this loop interact with ATP via the gamma phosphate groups and these interactions are conserved among mTOR and PI3Ks. In mTOR, the P-loop has a conserved serine, which coordinates the beta-phosphate of ATP that is also seen in PI3Ks (Fig. 6). Moreover, mTOR also has another conserved residue, lysine that is covalently modified by wortmannin in mTOR and the PI3Ks.  The amphipathic  helix serves as basal element of the active site in the protein kinases. In the mTOR, the hydrophilic side of the  is exposed toward the ATP-binding site residues and the FAT domain while the hydrophobic side packs with helices 6/8/9 (Fig. 6). In the PI3Ks, the helix  makes equivalent interactions with the ATP-binding site, 8/9 and the helical domain Gln711-Gln400. 

The C-lobe of the mTOR contains the majority of the active site. The mTOR activation loop has two conserved motifs at the N-lobe and the C-lobe, HIDFG with the highly conserved DFG motif is present at the N-terminal end of the activation loop of both PI3Ks. From the structure of the PI3Ks (Fig. 6), the catalytic and the activation loops are locked in different conformations. In the recent crystal structure of mTOR complex bound to ADP-Mg-F (Fig. 6), the conserved residues of both DRHN and the DFG motifs are positions toward the active site, which indicates that the mTOR is an intrinsically active enzyme. For the PI3K family, no equivalent change in conformation has been observed and they are not regulated by activation loop phosphorylation.

The FATC has and a conserved among the mTOR orthologous at the end of the C-lobe (Fig. 6). The FATC is stabilized through interactions with the active loop on one side and a hydrophobic interface with the LBE on the other side. The loop uses the C-terminal motif to clamp the hinge of FATC onto the LBE and where the  bond to mLST8. In this way the activation loop forms a cage structure around the FATC hinge, stacking its phenylalanine perpendicular to the tyrosine, which helps to position hydrophobic patch of  toward the active site.  The structural organization of the mTOR helices , , and  resembles the C-terminal regulatory arch composed of the equivalent helices in PI3Ks (Fig. 6). In PI3Ks, helix  sits on the surface of the C-lobe where it reflect an active to inactive sate transition while in the mTOR it is hidden behind the LBE and it does not seem to be involved in an open-to closed transition. Moreover, the mTOR structure and in vitro kinase activity suggest that the conformation of the kinase domain with the folded inside the C-lobe is inherently open and active in the absence of the regulatory subunits such as he RAPTOR and RICTOR/mSIN1. Furthermore, the helices , and  together with C-terminal regulatory arch in mTOR contains a unique fourth helix b that is conserved among mTOR orthologues and not present in PI3Ks. The b helix packs against the activation loop, where it caps the catalytic site and overlaps with the negative regulatory domain (RD is a region of residues between 2430-2450). A deletion of the RD causes an increase of the mTOR kinase activity, but that is believed is due to b since its removal decreases mTOR kinase activity. One theory suggest that the shortening the linker between helices b and  causes tighter packing of the two helices with the activation loop which limits accessibility to the active site. As in P (Fig.6), a number of the mTOR activating mutations are found along the regulatory arch, thus it is believed that Ras homolog enriched in the brain (RHEB) activates mTOR by interacting with the kinase domain active site, mLST8, and RAPTOR.

·      The ATP-binding pocket and the catalytic loop.

The  ,a highly conserved sequence motif, is found in the active site of the mTOR kinase domain (Fig. 7a).  The lysine  residue makes interaction with the alpha and /or the beta of the phosphate group of ATP.  The residues following the lysine () forms a connecting loop between  and  marked by RQD residues. The  loop residues contribute to the ATP-binding pocket. The Glycine 2188 residue allows for the close packing of the first helix of kinase (). Histidine 2189 donates hydrogen bonds to a conserved residue in the ATP-loop, glutamine 2167. Aspartic acid 2191 contributes to the overall orientation of the GHEDL loop as well as provides polar contact for the side chain arginine 2193 that is located at the N-terminal end of . Leucine 2192 provides a binding site for the  strand, the residues of which form the hydrophobic pocket for the adenine ring.  helix is important for the organization of the kinase domain. It is located at the beginning of the C-lobe (Fig. 6). The first three residues, , are centered at the kinase domain and tend to interact with residues involved in ATP-binding site. The arginine 2193 is involved in the orientation of the Aspartic acid 2191in the GHEDL loop. Moreover the aspartic acid 2195 contacts the backbone nitrogen of the activation loop phenylalanine 2358, which is involved in the stacking platform for  Recently, this motif has been shown to play an important role as a binding surface for the  helix.

The catalytic loop has a signature motif made of . The homology model found that the  triplet is always followed by Proline 2341, which defines a unique feature of the TOR family (Fig. 8 blue).  Pro2341 forms a hydrophobic core at the C-terminal end of the catalytic loop. It is centered on Trp2549, the last residue of mTOR. The mTOR have three important loops that are vital for its catalytic activity: the activation loop, the catalytic loop, and the P-loop. The activation loop is part of the polypeptide-binding site, which carried the DFG motif with the Asp interacting with the cofactor  at the active site. the catalytic loop carries three important residues, DHN, that area involved in the catalytic reaction. Aspartic acid is involved in the orientation of the substrate as well as the polarization of the hydroxyl group. The histidine is involved in stabilizing the gamma phosphate transition state and the asparagine stabilizes the second metal ligand.

·      FRB role in mTOR kinase domain.

Acute rapamycin treatments inhibit the catalytic activity and signaling capacity of mTORC1 while it fails to inhibit mTORC2. The rapamycin-binding site maps to the FRB surface closest to the active site, suggesting that the rapamycin-binding site interacts with substrates to facilitate their entry to the active site. Also, S6K1 and 4EBP1 contain a TOR signaling (TOS) motif that mediates essential interaction with the scaffolding protein raptor to facilitate the recruitment of substrates to the mTOR kinase. In order to map the region of S6K1 that is involved in FRB interactions, that region was deleted which led to the reducing of Thr 389 phosphorylation. The data obtained from that experiment indicated that the FRB provides a secondary substrate-recruitment site near the entrance of the catalytic cleft and they presumed that, although TOS motif, is the primary means of substrate recruitment, the secondary site may also facilitates substrate entry into the restricted active site as well as it can provide more specificity for the substrates. Furthermore, Rapamycin inhibits mTOR activity in a substrate and phosphorylation-site dependent manner. The tertiary complex, which consists of the rapamycin, FKBP12, and other conserved residues of the mTOR FRB domain forms just in front of the catalytic cleft, which constrict access to the active site. It is also suggested that the rapamycin binds to a conserved secondary substrate site on the FRB meaning that rapamycin is actually a competitive inhibitor for the protein substrate. 

·      Structure of mTORC1 and mTORC2:

 

III.         Biology and biological implications:

 

·      Cellular signaling of mTORC1:

mTOR interacts with different proteins to form at least two functional signaling complexes, mTORC1 and mTORC2. At the cellular level, mTORC1 promotes cellular anabolic processes, including protein synthesis, cell growth, ribosome biogenesis, and cell cycle progression (Fig.9). During growth factor and nutrient sufficiency, mTORC1 directly phosphorylates S6K1 and eIF4E to coordinate protein synthesis. Consequently, S6K1 phosphorylates and activates several substrate that promotes mRNA translation initiation including eIF4B (Fig. 4b). mTORC1 phosphorylate 4EBP to trigger its dissociation from The 5′-cap-binding protein eLF4E, which allows mRNA translation to occur (Fig. 4b). Furthermore, the global ribosome footprinting analyses discovered that mTOR inhibition profoundly affects mRNAs containing pyrimidine –rich 5′ TOP motif, which includes genes that are involved in protein synthesis. mTORC1 also facilitates growth by promoting a shift in glucose metabolism from oxidative pentose phosphate pathway (PPP), which utilizes carbons from glucose to generate NADPH and other intermediary metabolites needed for proliferation and growth (Fig. 4b). mTORC1 is also involved in protein turnover. It does this by suppressing protein catabolism and most importantly inhibits autophagy by phosphorylating and inactivation the authophagic protein ULK1 (Fig. 4a). When the cell is under nutrient replete conditions, mTORC1 phosphorylates ULK1, which leads to the inhibition of mTORC1 by the AMP-activated protein kinase (AMPK) (Fig. 9). There is also another major pathway responsible for protein turnover, the ubiquitin-proteasome system (UPS) that tag proteins and targeted them for degradation. Recent studies found that acute mTORC1 inhibition increases proteolysis through either increase in protein ubiquitylation, or an increased abundance of protosamal chaperons via inhibition of Erk5 (Fig 4a). Also, there have been some explanation that while acute inhibition of mTORC1 promotes proteolysis, the prolonged mTORC1 activation also increase in protein turnover to balance the increased rate of protein synthesis and maintain homeostasis in the cell. Extensive research effort has been dedicated to identify the link environmental cues to mTORC1 regulation. The mTORC1-inhibitor tuberous sclerosis complex (TSC) is an important central complex that regulate mTORC1 (Fig. 9).  TSC is composed of Tsc2 and Tsc1 and the inactivation of either one will lead to strong mTORC1 signaling. Tsc2 contains a GTP-activating protein (GAP) domain that acts on Rheb leading to the activation of mTORC1 via an incompletely defined mechanism that is still unknown. In addition, the insulin/phosphatidylinositol 3-kinase (PI3K) signaling promotes Akt-mediated phosphorylation of Tsc2, which suppress the inhibitory effect of Tsc1 and Tsc2 on mTORC1 and that activated Rheb (Fig.9).

·      Cellular signaling of mTORC2:

Most of the attention is focused on mTORC1 due to its ample amount of pathways and activation/inhibition loops, however recent research efforts are being contributed to the role of mTORC2. While mTROC1 promotes cellular anabolic process, mTORC2 controls proliferation and survival mainly by phosphorylating many members of the AGC family of protein kinases. mTORC2 most important downstream regulation is to phosphorylate and activate protein kinase B also known as Akt (Fig.9). Akt is a serine/threonine specific protein kinase, which plays an important key in insulin/PI3K signaling. Activated Akt plays multiple roles in cellular processes like glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration (Fig 4c). In addition of activating many cellular pathways, Akt also inhibits several substrates like FoxO1/3a (Fig. 4c) transcription factors and the mTORC1 inhibitor Tsc2 (Fig. 9). Moreover, mTORC2 is also involved in the phosphorylation and activation of SGK1 (Fig. 4c). SGK1another AGC-kinase is involved in the regulation of a wide variety of ion channels, membrane transporters, cellular enzymes, transcription factors, migration and apoptosis. Upstream regulation of mTORC2 regulated by mTORC1 because of the presence of the negative feedback loop between mTORC1 and the insulin/PI3K signaling (Fig. 9). Also, Sk61 suppress mTORC2 activation via the phosphorylation-dependent degradation of insulin receptor substrate 1 (IRS1) (Fig.9). Indeed this negative feedback regulation of PI3K and mTORC2 signaling by mTORC1 has many implications for the pharmacological targeting of mTORC1 diseases.