Stress-induced phosphoprotein 1 (STIP1, also known as HSP70-HSP90-organizing protein or HOP) functions as a mediator of interaction between heat shock protein (HSP)70 and HSP90 as part of the cellular assembly machine. It also modulates the ATPase activity of both HSP70 and HSP90, thus facilitating client protein transfer between the two. STIP1 is a monomeric protein composed of three tetratricopeptide repeat domains (TPR1, TPR2A, TPR2B) involved in protein-protein interactions, and two small aspartic acid–proline repeat domains (DP1, DP2) involved in client activation (Scheufler C et al. 2000; Nelson GM et al. 2003; Yi F et al. 2010; Schmid AB et al. 2012). A flexible linker of STIP1 (HOP) connects TPR1-DP1 and TPR2A-TPR2B-DP2 modules arranging it as TPR1-DP1-TPR2A-TPR2B-DP2 (Scheufler C et al. 2000). Biochemical and crystallographic analysis revealed that TPR domains of STIP1 interact specifically with C-terminal MEEVD motifs of HSP70 or HSP90 chaperones; TPR2A binds preferentially to HSP90, whereas TPR1 and TPR2B bind to HSP70 (Scheufler C et al. 2000; Carrigan PE et al. 2006; Schmid AB et al. 2012). Furthermore, cryoelectron microscopy (cryo-EM) reconstruction of the human HSP90:STIP1 complex revealed that STIP1 may also form interactions in several other parts of HSP90, pre-organizing N-terminal domains (NTDs) of HSP90 and thus increasing accessibility of the nucleotide-binding pocket (Southworth DR & Agard DA 2011). STIP1 stabilizes an alternate HSP90 open state where hydrophobic client-binding surfaces of HSP90 monomers have converged remaining accessible for client loading (Southworth DR & Agard DA 2011). STIP1 is positioned with a TPR1 domain extending from the HSP90 dimer cleft remaining available for an interaction with HSP70. In the STIP1-stabilized HSP90 conformation the N-terminal domains have rotated to match the closed ATP conformation. However, the arrangement of the STIP1 domains in the complex seems to prevent the NTDs dimerization of HSP90 monomers and total closure of the HSP90 dimer that is required for an efficient HSP90-mediated ATP hydrolysis (Southworth DR & Agard DA 2011; Alvira S et al. 2014). HSP70, in the ADP state, readily binds HSP90:STIP1, forming a client-loading complex HSP90:STIP1:HSP70:client protein (Hernández MP et al. 2002). Structural studies of GR-LBD (the ligand-binding domain of the glucocorticoid receptor) bound to HSP90:STIP1:HSP70 complex showed that one STIP1 molecule binds to the HSP90 dimer and through domain rearrangement, gives rise to two main conformations, an extended structure that recognizes and interacts with HSP70, and a compact one in which HSP70 is in contact with one HSP90 monomer (Alvira S et al. 2014). Movement between these two modes is thought to deliver the HSP70-bound substrate to the side of the HSP90 dimer opposite the site of STIP1 binding (Alvira S et al. 2014). Following client delivery by HSP70 and STIP1 release, HSP90:ATP converts to the closed ATP hydrolysis-active state to complete the chaperone cycling.

Immunophilin p23 (also known as PTGES3) binds selectively to the ATP-bound state of HSP90. p23 stabilizes the closed state of HSP90, which weakens the binding of STIP1(HOP) and promotes its exit from the complex (McLaughlin H et al. 2006; Karagöz GE et al. 2011). When FKBP51 (FKBP5) is present, a stable intermediate FKBP51:GR:HSP90:p23 is formed by expulsion of HSP70 and STIP1(HOP) (Ebong I et al. 2016).

Mass spectrometry analysis showed that FKBP51 (FKBP5) and FKBP52 (FKBP4) form analogous complexes with GR:HSP90:STIP1:HSP70:ATP (Ebong IO et al. 2016). Without hormone, FKBP51 is the major immunophilin in GR:HSP90 complexes, whereas after hormone treatment, FKBP52 rapidly replaces FKBP51 (Davies et al., 2002).

FK506 binding protein 5 (FKBP51, also known as FKBP5) is a member of the immunophilin (IMM) protein family of intracellular proteins. The signature domain of the IMM family is the peptidyl-prolyl-cis/trans-isomerase (PPIase) domain, which is in turn the drug binding domain. IMMs are classified by their ability to bind immunosuppressant drugs – CyPs (cyclophilins) bind cyclosporine A (CsA), and FKBPs (FK506-binding pro-teins) bind FK506 (Pratt and Toft 1997; Kang et al. 2008). In addition to the PPIase domain, there are three additional domains – the nucleotide-binding domain, (also called FKBD2 in FKBP proteins) where ATP binds, the calmodulin-binding domain, a poorly characterized domain able to interact with calmodulin, and tetratricopeptide repeat (TPR) domains, sequences of 34 amino acids repeated in tandem through which FKBPs bind to the HSP90 C-terminal sequence MEEVD (Davies et al. 2005; Wu et al. 2004). Mass spectrometry analysis showed that FKBP51 (FKBP5) and FKBP52 (FKBP4) form analogous complexes with GR:HSP90:STIP1:HSP70:ATP (Ebong IO et al. 2016). Binding of FKBP51 (FKBP5) and other immunophilins may weaken the association of TPR domain containing protein STIP1 with HSP90 complex (Li et al. 2011).

Immunophilin p23 (also known as PTGES3) binds selectively to the ATP-bound state of HSP90. p23 stabilizes the closed state of HSP90, which weakens the binding of STIP1(HOP) and promotes its exit from the complex (McLaughlin H et al. 2006; Karagöz GE et al. 2011). When p23 is added to the client-transfer complex in the absence of the immunophilin or with FKBP51 (FKBP5), two copies of p23 are incorporated with concomitant loss of HSP70 and HOP (Ebong I et al. 2016). By contrast no stable complex with two p23 subunits is observed in the presence of FKBP52 (FKBP4); expulsion of HSP70, HOP and p23 occur with a low population of a complex incorporating only one p23 subunit (Ebong I et al. 2016).

FKBP52 (also known as the large immunophilin FKBP4) is a co-chaperone containing tetratricopeptide repeat (TPR) domain, which binds the C-terminal sequence motif (MEEVD) of HSP90 (Wu B et al. 2004; Davies and Sanchez 2005). The stoichiometry of FKBP in receptor heterocomplexes was determined on the basis of the size of cross-linked complexes, a ratio of one molecule of receptor and two molecules of HSP90 to one molecule of FKBP52 was obtained for human PR , ER and mouse GR (Rexin M et al. 1992; Rehberger P et al. 1992; Segnitz B and Gehring U 1995). Mass spectrometry analysis showed that FKBP51 (FKBP5) and FKBP52 (FKBP4) form analogous complexes with GR:HSP90:STIP1:HSP70:ATP (Ebong IO et al. 2016). Binding of FKBP52 (FKBP4) and other immunophilins may weaken the association of TPR domain containing protein STIP1 with HSP90 complex (Li et al. 2011).

FKBP52 (FKBP4) is a member of the immunophilin (IMM) protein family of intracellular proteins that are able to bind immunosuppressant drugs, from which the term immunophilin derives (Pratt and Toft 1997; Kang et al. 2008). These proteins are also known as peptidyl-prolyl cis/trans isomerases (PPIases) for their ability to convert proline bonds from cis to trans form, a rate-limiting step in protein folding (Harding et al. 1989; Standaert et al. 1990; Galat 2003; Davies and Sanchez 2005). In addition to the PPIase and TPR domains, there are two additional domains - the nucleotide-binding domain (also called FKBD2 in FKBP proteins) where ATP binds and the calmodulin-binding domain, a poorly characterized domain able to interact with calmodulin.

Receptor-interacting serine/threonine protein kinase 3 (RIPK3) functions as a key regulator of necroptosis.The protein stability of RIPK3 is negatively regulated by the C-terminus of HSC70-interacting protein (CHIP, also known as STIP1 homology and U-Box containing protein 1, STUB1) (Seo J et al. 2016). STUB1, as an E3 ligase, mediates ubiquitylation of RIPK3 at Lys55 and Lys363 and targerts it to lysosomal degradation. Coimmunoprecipitation analysis using overexpressed proteins revealed interactions between STUB1 (CHIP) and RIPK1 in human embryonic kidney 293T (HEK293T) cells (Seo J et al. 2016). Interactions of endogenous STUB1 and RIPK1 proteins was detected in mouse fibroblast L929 cells. Direct interaction between recombinant proteins was confirmed by GST-pull-down assay (Seo J et al. 2016). Domain mapping revealed that the kinase domain of RIPK3 interacts with the tetratricopeptide repeat (TPR) region of STUB1 (Seo J et al. 2016). Treatment with geldanamycin (an inhibitor of HSP90) induced the degradation of RIPK3 in mouse fibroblasts L929 cells even under STUB1-depleted conditions, suggesting that HSP90 might not be involved in the STUB1-mediated degradation of RIPK3 (Seo J et al. 2016).

The chaperoning function of HSP90 is coupled to its ATPase activity. Our current understanding of the ATPase mechanism of Hsp90 is based largely on structural and functional studied for the Saccharomyces cerevisiae Hsp90 complexes (Meyer P et al. 2003, 2004; Ali MM et al. 2006; Prodromou C et al. 2000; Prodromou C 2012). The ATPase cycle of human HSP90 is less well understood, however several studies suggest that the underlying enzymatic mechanisms and a set of conformational changes that accompany the ATPase cycle are highly similar in both species (Richter K et al. 2008; Vaughan CK et al. 2009). Once ATP is bound it helps to stabilize the closed ATP lid state, in which the gamma-phosphate of ATP provides a hydrogen bonding that promotes a stable association of the ATP lid with N-terminal domain (NTD) (Ali MM et al. 2006; Prodromou C et al. 2000; Chadli A et al. 2000). The association of ATP with NTD then stimulates structural changes in NTD and in the middle domain that are likely to involve movements of the ATP lid segment within each N-terminal domain that locates over the bound ATP. The movement of the lids exposes surface residues that are subsequently involved in transient dimerization of the N-terminal domains of HSP90 (Ali MM et al. 2006; Prodromou C et al. 2000; Chadli A et al. 2000). Furthermore, the intrachain associations of NTD with the middle domain leads to the active conformation of the catalytic loop of HSP90, which commits the ATP for hydrolysis (Meyer P et al. 2003). The subsequent conformational changes upon ATP binding are regulated by co-chaperone activities. For example, arrangement of the STIP1 domains in the complex seems to prevent the NTDs dimerization of HSP90 monomers and total closure of the HSP90 dimer that is required for an efficient HSP90-mediated ATP hydrolysis (Southworth DR & Agard DA 2011; Alvira S et al. 2014). In addition, client protein binding to HSP90 was found to increase ATPase activity of HSP90 up to 200-fold (McLaughlin SH et al. 2002).

After hydrolysis of ATP the ligand-bound steroid hormone receptor (SHR) is released from HSP90 complex. The Reactome module describes ATPase activity of HSP90 in the nucleus, however it is not entirely clear whether cytosolic hormone-bound SHR translocates through the nuclear pores before or after ATP-dependent dissociation from the HSP90 complex.

The binding of TNF-α to TNF receptor 1 (TNFR1) results in the sequential formation of several signaling complexes (Walczak H 2011). The rapidly forming complex-I (TNFR1 signaling complex) is assembled at the receptor’s cytoplasmic tail and consists of TNFR1, TRADD (TNFR1-associated death domain), TRAF2 (TNF receptor associated factor-2), RIPK1 (receptor-interacting serine/threonine protein kinase 1), and E3 ubiquitin (Ub) ligases BIRC2, BIRC3 (cIAP1/2, cellular inhibitor of apoptosis) and LUBAC (linear ubiquitin chain assembly complex) (Micheau O and Tschopp J 2003; Yuan J et al. 2019). Within this complex, RIPK1 and other proteins are rapidly conjugated with Ub chains by various E3 ligases (Micheau O and Tschopp J 2003; Yuan J et al. 2019). The ubiquitination status of RIPK1 determines cell fate downstream of the TNFR1 signaling complex (Yuan J et al. 2019). The conjugation of K63-linked Ub chains by BIRC2/3 or Met1-linked Ub chains by LUBAC, have been shown to promote RIPK1-dependent pro-survival NF-kappa-B signaling while inhibiting RIPK1 kinase-mediated apoptosis and necroptosis. In addition, RIPK1 also interacts with FADD/Caspase-8 or RIPK3/MLKL to form complex IIa or IIb, which activate apoptosis or necroptosis respectively (Yuan J et al. 2019). In these cell death-inducing complexes, RIPK1 function is also regulated by ubiquitination (Amin P et al. 2018; de Almagro MC et al. 2015). The protein stability of RIPK1 is negatively regulated by the carboxyl-terminus of HSC70-interacting protein (CHIP, also known as STIP1 homology and U-Box containing protein 1, STUB1) (Seo J et al. 2016). STUB1 (CHIP), as an E3 ligase, mediates K48-linked ubiquitination of RIPK1 at K571, K604, and K627 and targets it to lysosomal degradation (Seo J et al. 2016). Co-immunoprecipitation analysis using overexpressed proteins revealed interactions between STUB1 (CHIP) and RIPK1 in human embryonic kidney 293T (HEK293T) cells (Seo J et al. 2016). Interaction of endogenous STUB1 and RIPK1 proteins was detected in mouse fibroblast L929 cells. Direct interaction between recombinant proteins was confirmed by GST-pull-down assay (Seo J et al. 2016). Domain mapping revealed that the RHIM domain of RIPK1 interacts with the U-Box domain of STUB1 (Seo J et al. 2016). Similarly, STUB1 interacts with and ubiquitinates RIPK3 inducing lysosome-dependent destabilization of RIPK3 (Seo J et al. 2016). These data suggest that the E3 ligase activity of STUB1 negatively regulates TNF-α–induced cell death by conjugating K48-linked ubiquitin chains to RIPK1 and RIPK3, thereby promoting their degradation.

This Reactome event describes STUB1 (CHIP) binding to RIPK1.

The molecular chaperone heat-shock protein 90 (HSP90) functions as a homodimer. Each HSP90 protomer contains three flexibly linked regions, the N-terminal ATP-binding domain (NTD), the middle domain, and the C-terminal dimerization domain (Prodromou C et al. 1997; Pearl LH and Prodromou C 2006). HSP90 dimer is rather a dynamic molecule and ATP binding and hydrolysis are associated with conformational changes (Obermann WM et al. 1998; Krukenberg KA et al. 2011; Li J & Buchner J 2013; Prodromou C 2012). The structures of the isolated yeast and human N-terminal domain (NTD) of HSP90 bound to ATP, ADP and adenylylimidodiphosphate (AMP-PNP, a non-hydrolysable analogue of ATP) suggest that nucleotides bind deep in the cleft of NTD in open apo state of HSP90 (Prodromou C et al.1997; Meyer P et al. 2003, 2004; Colombo G et al. 2008; Li J et al. 2012). The structural studies of NTD of human HSP90 with antitumor agent geldanamycin (that acts as an ADP/ATP mimetic) support the polar interactions in the binding pocket described for yeast Hsp90 and ADP or ATP (Stebbins CE et al. 1997; Prodromou C et al.1997; Grenert JP et al. 1997). Once ATP is bound it helps to stabilize the closed ATP lid state, in which the gamma-phosphate of ATP provides a hydrogen bonding that promotes a stable association of the ATP lid with NTD. The association of ATP or AMP-PNP with NTD then stimulates structural changes in NTD. NMR analysis of human full-length HSP90 protein with and without ATP confirmed that ATP binding led to conformational changes in NTD (Karagöz GE et al. 2010). No structural changes were observed in the middle and C-terminal domains (Karagöz GE et al. 2010). However, other studies suggest that ATP-dependent conformational changes occur both in NTD and in the middle domain of HSP90 (Ali MM et al. 2006; Prodromou C et al. 2000; Chadli A et al. 2000; Meyer P et al. 2003). The changes are likely to involve movements of the ATP lid segment within each N-terminal domain that locates over the bound ATP (Ali MM et al. 2006; Prodromou C et al. 2000; Chadli A et al. 2000). The movement of the lids exposes surface residues that are subsequently involved in transient dimerization of the N-terminal domains of HSP90 (Ali MM et al. 2006; Prodromou C et al. 2000; Chadli A et al. 2000). The subsequent conformational changes upon ATP binding are regulated by co-chaperone activities. For example, arrangement of the STIP1 domains in the complex seems to prevent the NTDs dimerization of HSP90 monomers and total closure of the HSP90 dimer that is required for an efficient HSP90-mediated ATP hydrolysis (Southworth DR & Agard DA 2011; Alvira S et al. 2014). Thus, ATP binding coupled to co-chaperone-mediated loading of client protein to HSP90 complex regulates ATPase activity of HSP90.

Active GTP-bound RND1 binds the following effectors:
ARHGAP5 (Wennerberg et al. 2003; Bagci et al. 2020)
FRS2 (Harada et al. 2005)
FRS3 (Harada et al. 2005)
GRB7 (Vayssiere et al. 2000)
PLEKHG5 (Goh and Manser 2010)
PLXNA1 (Zanata et al. 2002)
STIP1 (de Souza et al. 2014)
STMN2 (Li et al. 2009)
UBXN11 (Katoh et al. 2002)

The following candidate RND1 effectors were reported in the high throughput screen by Bagci et al. 2020 or have been reported as RND1 effectors in some but not all studies:
ALDH3A2 (Bagci et al. 2020)
ANKRD26 (Bagci et al. 2020)
ARHGAP35 (Wennerberg et al. 2003, Mori et al. 2009: binding to active RND1; Bagci et al. 2020: no binding to active RND1)
CAV1 (Bagci et al. 2020)
CCDC88A (Bagci et al. 2020)
CPD (Bagci et al. 2020)
DEPDC1B (Bagci et al. 2020)
DLG5 (Bagci et al. 2020)
DSP (Bagci et al. 2020)
DST (Bagci et al. 2020)
EPHA2 (Bagci et al. 2020)
EPSTI1 (Bagci et al. 2020)
FAM135A (Bagci et al. 2020)
FAM83B (Bagci et al. 2020)
FLOT2 (Bagci et al. 2020)
KIDINS220 (Bagci et al. 2020)
KIF14 (Bagci et al. 2020)
LEMD3 (Bagci et al. 2020)
MUC13 (Bagci et al. 2020)
PKP4 (Bagci et al. 2020)
PIK3R1 (Bagci et al. 2020)
PIK3R2 (Bagci et al. 2020)
PTPN13 (Bagci et al. 2020)
RASAL2 (Bagci et al. 2020)
RBMX (Bagci et al. 2020)
RRAS2 (Bagci et al. 2020)
TFRC (Bagci et al. 2020)
TMEM59 (Bagci et al. 2020)
TXNL1 (Bagci et al. 2020)
VANGL1 (Bagci et al. 2020)
VANGL2 (Bagci et al. 2020)
WDR6 (Bagci et al. 2020)

RND1 does not interact with the following putative effectors that bind to active RND2 and/or RND3:
CKAP4 (Bagci et al. 2020)
CKB (Bagci et al. 2020)
DDX4 (Bagci et al. 2020)
DSG1 (Bagci et al. 2020)
FNBP1 (Bagci et al. 2020)
GOLGA3 (Bagci et al. 2020)
KTN1 (Bagci et al. 2020)
LRRC1 (Bagci et al. 2020)
NISCH (Bagci et al. 2020)
NUDC (Bagci et al. 2020)
PICALM (Bagci et al. 2020)
SCRIB (Bagci et al. 2020)
SEMA4F (Bagci et al. 2020)
TMOD3 (Bagci et al. 2020)
UHRF1BP1L (Bagci et al. 2020)

Active GTP bound RND2 binds the following effectors:
ARHGAP5 (Wennerberg et al. 2003; Bagi et al. 2020)
FNBP1 (Fujita et al. 2002; Kakimoto et al. 2004; Bagci et al. 2020)
FRS2 (Harada et al. 2005)
FRS3 (Harada et al. 2005)
KCTD13 (Gladwyn Ng et al. 2016)
PLXND1 (Uesugi et al. 2009)
PRAG1 (Tanaka et al. 2006)
TNFAIP1 (Gladwyn Ng et al. 2015; Gladwyn Ng et al. 2016)
UBXN11 (Katoh et al. 2002)

RND2 binds to the following candidate effectors reported in the high throughput screen by Bagci et al. 2020 or reported in some but all studies:
ALDH3A2 (Bagci et al. 2020)
ANKRD26 (Bagci et al. 2020)
ARHGAP1 (Bagci et al. 2020)
ARHGAP35 (Wennerberg et al. 2003: binding to active RND2; Bagci et al. 2020: no binding to active RND2)
CAV1 (Bagci et al. 2020)
CKAP4 (Bagci et al. 2020)
DEPDC1B (Bagci et al. 2020)
DLG5 (Bagci et al. 2020)
DSG1 (Bagci et al. 2020)
DST (Bagci et al. 2020)
EPHA2 (Bagci et al. 2020)
FAM83B (Bagci et al. 2020)
GOLGA3 (Bagci et al. 2020)
KIDINS220 (Bagci et al. 2020)
KIF14 (Bagci et al. 2020)
KTN1 (Bagci et al. 2020) - while RND2 has not been shown to localize to the endoplasmic reticulum membrane (ER), some isoforms of KTN1 are known to localize to the plasma membrane instead of the ER membrane (Santama et al. 2004)
LEMD3 (Bagci et al. 2020)
LRRC1 (Bagci et al. 2020)
MUC13 (Bagci et al. 2020)
NISCH (Bagci et al. 2020)
NUDC (Bagci et al. 2020)
PIK3R1 (Bagci et al. 2020)
PIK3R2 (Bagci et al. 2020)
PKP4 (Bagci et al. 2020)
PTPN13 (Bagci et al. 2020)
RBMX (Bagci et al. 2020)
SCRIB (Bagci et al. 2020)
TFRC (Bagci et al. 2020)
TXNL1 (Bagci et al. 2020)
UHRF1BP1L (Bagci et al. 2020)
VANGL1 (Bagci et al. 2020)
VANGL2 (Bagci et al. 2020)
WDR6 (Bagci et al. 2020)

RND2 does not bind the following effectors:
CCDC88A (Bagci et al. 2020)
CKB (Bagci et al. 2020)
CPD (Bagci et al. 2020)
DDX4 (Bagci et al. 2020)
DSP (Bagci et al. 2020)
EPSTI1 (Bagci et al. 2020)
FAM135A (Bagci et al. 2020)
FLOT2 (Bagci et al. 2020)
GRB7 (Vayssiere et al. 2000)
PLEKHG5 (Goh and Manser 2010)
PICALM (Bagci et al. 2020)
RASAL2 (Bagci et al. 2020)
RRAS2 (Bagci et al. 2020)
SEMA4F (Bagci et al. 2020)
STIP1 (de Souza et al. 2014)
STMN2 (Li et al. 2009)
TMEM59 (Bagci et al. 2020)
TMOD3 (Bagci et al. 2020)

Carboxyl-terminus of HSC70-interacting protein (CHIP, also known as STIP1 homology and U-Box containing protein 1, STUB1) is a cochaperone E3 ligase. STUB1 (CHIP) contains three tandem repeats of tetratricopeptide (TPR) motifs and a C-terminal U-box domain separated by a charged coiled-coil region (Paul I & Ghosh MK 2014). STUB1 functions as a negative co-chaperone for the HSP90/HSP70 chaperone to regulate protein quality control by targeting unfolded or misfolded proteins for proteasomal degradation. Structural studies suggest that STUB1 functions as a homodimer (Zhang M et al. 2005). In addition, STUB1 (CHIP) also targets many mature proteins for ubiquitination and degradation or degradation-independent regulation (Paul I & Ghosh MK 2014). STUB1 has been shown to affect apoptotic cell death by negatively regulating a variety of tumor suppressive factors (Ahmed SF et al. 2012; Esser C et al. 2005). STUB1 (CHIP) has been also implicated in down-regulation of necroptosis via targeting receptor-interacting serine/threonine protein kinase 1 (RIPK1) and RIPK3 (Seo J et al. 2016). STUB1 deficiency in mouse embryonic fibroblasts (MEF), mouse fibroblasts (L929) and human colorectal adenocarcinoma (HT-29) cells exhibited higher levels of RIPK1 and RIPK3 expression, resulting in increased sensitivity to necroptosis induced by TNFα (Seo J et al. 2016). Supporting these findings, in vivo studies demonstrated that the inflammatory and lethal phenotypes of Chip−/− mice were rescued by crossing with Ripk3 knockout mice (Seo J et al. 2016). Coimmunoprecipitation analysis revealed interactions between STUB1 and RIPK1 or RIPK3 (Seo J et al. 2016). K571, K604, and K627 as ubiquitination lysine sites of RIPK1 were detected by mass spectrometry (Mollah S et al. 2007; Kim W et al. 2011). Mutagenesis analyses of RIPK1 demonstrated that STUB1 (CHIP)-mediated K48-ubiquitination on K571, K604, and K627 of RIPK1 is essential for the lysosome co-localization and degradation of RIPK1 upon co-expression of tagged proteins in human non–small-cell lung cancer H1299 cells (Seo J et al. 2016). Similarly, STUB1 ubiquitinated RIPK3 inducing lysosome-dependent destabilization of RIPK3 (Seo J et al. 2016). Further, RIPK1 I539D and RIPK3 V460P, that do not form a hetero-oligomeric amyloid signaling RIPK1:RIPK3 complex, also showed lysosomal localization upon ectopic expression of STUB1 in H1299 cells suggesting that STUB1 targets nascent forms of RIPK3 and RIPK1. The data suggest that STUB1 negatively regulates RIPK1 & RIPK3-mediated necroptosis via K48-linked ubiquitinitaion and lysosome-dependent destabilization of RIPK1 and RIPK3.

RIPK1 functions as a key regulator of the TNF receptor 1 (TNFR1) signaling, which is activated upon binding of TNF-α to TNF receptor 1 (TNFR1). Activation of TNFR1 results in the sequential formation of several signaling complexes (Micheau O and Tschopp J 2003; Walczak H 2011). The rapidly forming complex-I (the TNFR1 signaling complex) is assembled at the receptor’s cytoplasmic tail and consists of TNFR1, TRADD (TNFR1-associated death domain), TRAF2 (TNF receptor associated factor-2), RIPK1, and E3 ubiquitin (Ub) ligases BIRC2, BIRC3 (cIAP1/2, cellular inhibitor of apoptosis) and LUBAC (linear ubiquitin chain assembly complex) (Micheau O and Tschopp J 2003; Yuan J et al. 2019). Within this complex, RIPK1 and other proteins are rapidly conjugated with Ub chains by various E3 ligases (Micheau O and Tschopp J 2003; Walczak H 2011; Yuan J et al. 2019; Roberts JZ et al. 2022). The ubiquitination/deubiquitination status of RIPK1 determines cell fate downstream of the TNFR1 signaling complex (Yuan J et al. 2019; Roberts JZ et al. 2022). The conjugation of K63-linked Ub chains by BIRC2/3 or Met1-linked Ub chains by LUBAC, have been shown to promote RIPK1-dependent pro-survival NF-kappa-B signaling while inhibiting RIPK1 kinase-mediated apoptosis and necroptosis. E3 ubiquitin ligase activity of MIB2 also protects cells from the RIPK1-mediated cell death (Feltham R et al. 2018). Deubiquitination of RIPK1 abolishes its ability to activate NF-kappa-B upon TNF-α stimulation and leads to the formation of the cytosolic complex IIa, TRADD:TRAF2:RIPK1:FADD:caspase-8, which activates apoptosis. In addition, RIPK1 also interacts with RIPK3 and MLKL to form complex IIb, which activates necroptosis (Micheau O and Tschopp J 2003; Yuan J et al. 2019). In these cell death-inducing complexes, RIPK1 activity is also regulated by ubiquitination (Amin P et al. 2018; de Almagro M et al. 2015). Finally, E3 ligase activity of STUB1 targets RIPK1 and RIPK3 for lysosome-dependent degradation suppressing necroptosis (Seo J et al. 2016).

This Reactome event describes STUB1-mediated K48-linked ubiquitination of RIPK1 at K571, K604, and K627 downstream of TNFR1.