Comparison of the computed three-dimensional structures of oncogenic forms (bound to GDP) of theras-Gene-Encoded p21 protein with the structure of the normal (non-transforming) wild-type protein

Theras-oncogene-encoded p21 protein becomes oncogenic if amino acid substitutions occur at critical positions in the polypeptide chain. The most commonly found oncogenic forms contain Val in place of Gly 12 or Leu in place of Gln 61. To determine the effects of these substitutions on the three-dimensional structure of the whole p21 protein, we have performed molecular dynamics calculations on each of these three proteins bound to GDP and magnesium ion to compute the average structures of each of the three forms. Comparisons of the computed average structures shows that both oncogenic forms with Val 12 and Leu 61 differ substantially in structure from that of the wild type (containing Gly 12 and Gln 61) in discrete regions: residues 10–16, 32–47, 55–74, 85–89, 100–110, and 119–134. All of these regions occur in exposed loops, and several of them have already been found to be involved in the cellular functioning of the p21 protein. These regions have also previously been identified as the most flexible domains of the wild-type protein and have been bound to be the same ones that differ in conformation between transforming and nontransforming p21 mutant proteins neither of which binds nucleotide. The two oncogenic forms have similar conformations in their carboxyl-terminal domains, but differ in conformation at residues 32–47 and 55–74. The former region is known to be involved in the interaction with at least three downstream effector target proteins. Thus, differences in structure between the two oncogenic proteins may reflect different relative affinities of each oncogenic protein for each of these effector targets. The latter region, 55–74, is known to be a highly mobile segment of the protein. The results strongly suggest that critical oncogenic amino acid substitutions in the p21 protein cause changes in the structures of vital domains of this protein.     

Structural effects of the binding of GTP to the wild-type and oncogenic forms of theras-gene-encoded p21 proteins

Molecular dynamics calculations have been performed to determine the average structures ofras-gene-encoded p21 proteins bound to GTP, i.e., the normal (wild-type) protein and two oncogenic forms of this protein, the Val 12- and Leu 61-p21 proteins. We find that the average structures for all of these proteins exhibit low coordinate fluctuations (which are highest for the normal protein), indicating convergence to specific structures. From previous dynamics calculations of the average structures of these proteins bound to GDP, major regional differences were found among these proteins (Monacoet al. (1995),J. Protein Chem., in press). We now find that the average structures of the oncogenic proteins are more similar to one another when the proteins are bound to GTP than when they are bound to GDP (Monacoet al. (1995),J. Protein Chem., in press). However, they still differ in structureat specific amino acid residues rather than in whole regions, in contradistinction to the results found for the p21-GDP complexes. Two exceptions are the regions 25–32, in an-helical region, and 97–110. The two oncogenic (Val 12- and Leu 61-) proteins have similar structures which differ significantly in the region of residues 97–110. This region has recently been identified as being critical in the interaction of p21 with kinase target proteins. The differences in structure between the oncogenic proteins suggest the existence of more than one oncogenic form of the p21 protein that can activate different signaling pathways.     

Comparison of the average structures,from molecular dynamics,of complexes of GTPase activating protein (GAP) with oncogenic and wild-type ras-p21: identification of potential effector domains

GTPase activating protein (GAP) is a known regulator of ras-p21 activity and is a likely target of ras-induced mitogenic signaling. The domains of GAP that may be involved in this signaling are unknown. In order to infer which domains of GAP may be involved, we have performed molecular dynamics calculations of GAP complexed to wild-type and oncogenic (Val 12–containing) ras-p21, both bound to GTP. We have computed and superimposed the average structures for both complexes and find that there are four domains of GAP that undergo major changes in conformation: residues 821–851, 917–924, 943–953, and 1003–1020. With the exception of the 943–953 domain, none of these domains is involved in making contacts with ras-p21, and all of them occur on the surface of the protein, making them good candidates for effector domains. In addition, three ras-p21 domains undergo major structural changes in the oncogenic p21-GAP complex: 71–76 from the switch 2 domain; 100–108, which interacts with SOS, jun and jun kinase (JNK); and residues 122–138. The change in conformation of the 71–76 domain appears to be induced by changes in conformation in the switch 1 domain (residues 32–40) and in the adjacent domain involving residues 21–31. In an accompanying paper, we present results from microinjection of peptides corresponding to each of these domains into oocytes induced to undergo maturation by oncogenic ras-p21 and by insulin-activated wild-type cellular p21 to determine whether these domain peptides may be involved in ras signaling through GAP.     

Comparison of the low energy conformations of an oncogenic and a non-oncogenic p21 protein, neither of which binds GTP or GDP

Oncogenic p21 protein, encoded by theras-oncogene, that causes malignant transformation of normal cells and many human tumors, is almost identical in sequence to its normal protooncogene-encoded counterpart protein, except for the substitution of arbitrary amino acids for the normally occurring amino acids at critical positions such as Gly 12 and Gin 61. Since p21 is normally activated by the binding of GTP in place of GDP, it has been postulated that oncogenic forms must retain bound GTP for prolonged time periods. However, two multiply substituted p21 proteins have been cloned, neither of which binds GDP or GTP. One of these mutant proteins with Val for Gly 10, Arg for Gly 12, and Thr for Ala 59 causes cell transformation, while the other, similar protein with Gly 10, Arg 12, Val for Gly 13 and Thr 59 does not transform cells. To define the critical conformational changes that occur in the p21 protein that cause it to become oncogenic, we have calculated the low energy conformations of the two multiply substituted mutant p21 proteins using a new adaptation of the electrostatically driven Monte Carlo (EDMC) technique, based on the program ECEPP. We have used this method to explore the conformational space available to both proteins and to compute the average structures for both using statistical mechanical averaging. Comparison of the average structures allows us to detect the major differences in conformation between the two proteins. Starting structures for each protein were calculated using the recently deposited x-ray crystal coordinates for the p21 protein, that was energy-refined using ECEPP, and then perturbed using the EDMC method to compute its average structure. The specific amino acid substitutions for both proteins were then generated into the lowest energy structure generated by this procedure, subjected to energy minimization and then to full EDMC perturbations. We find that both mutant proteins exhibit major differences in conformation in specific regions, viz., residues 35–47, 55–78, 81–93, 96–110, 115–126, and 123–134, compared with the EDMC-refined x-ray structure of the wild-type protein. These regions have been found to be the most flexible in the p21 protein bound to GDP from prior molecular dynamics calculations (Dykeset al., 1993). Comparison of the EDMC-average structure of the transforming mutant with that of the nontransforming mutant reveals major structural differences at residues 10–16, 32–40, and 60–68. These structural differences appear to be the ones that are critical in activation of the p21 protein. Analysis of the correlated motions of the different regions of the two mutant proteins reveals that changes in the conformation of regions in the carboxyl half of the protein are caused by changes in conformation around residues 10–16 and are transmitted by means of residues around Gln 61. The latter region therefore constitutes a molecular switch unit, in agreement with conclusions from prior work.On leave from the Department of Chemistry, University of Gdask, ul. Sobieskiego 18, 80-952 Gdask, Poland.     

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

Molecular dynamics analysis of the structures of ras-guanine nucleotide exchange protein (SOS) bound to wild-type and oncogenic ras-p21. Identification of effector domains of SOS

The X-ray crystal structure of the ras oncogene-encoded p21 protein bound to SOS, the guanine nucleotide exchange-promoting protein, has been determined. We have undertaken to determine if there are differences between the three-dimensional structures of SOS bound to normal and oncogenic (Val 12-p21) proteins. Using molecular dynamics, we have computed the average structures for both complexes and superimposed them. We find four domains of SOS that differ markedly in structure: 631–641, 676–691, 718–729, and 994–1004. Peptides corresponding to these sequences have been synthesized and found to be powerful modulators of oncogenic p21 in cells as described in an accompanying paper. We find that the SOS segment from 809–815 makes contacts with multiple domains of ras-p21 and can facilitate correlated conformational changes in these domains.     

Comparison of the predicted structure for the activated form of the P21 protein with the X-ray crystal structure

The predicted conformation and position of the central transforming region (residues 55–67) of the p21 protein are compared with the conformation and position of this segment in a recently determined X-ray crystal structure of residues 1–166 of this protein in the activated state bound to a nonhydrolyzable GTP derivative. We previously predicted that this segment of the protein would adopt a roughly extended conformation from Ile 55-Thr 58, a reverse turn at Ala 59-Gln 61, followed by an -helix from Glu 62-Met 67. We further predicted that this region of the activated protein occupies a position that is virtually identical to corresponding regions in the homologous purine nucleotide-binding proteins, bacterial elongation factor (EF-tu), and adenylate kinase (ADK). We find that there is a close correspondence between the conformation and position of our predicted structure and those found in the X-ray crystal structure. A mechanism for activation of the protein is proposed and is corroborated by X-ray crystallographic data.     

Comparison of the computed structures for the phosphate-binding loop of the p21 protein containing the oncogenic site Gly 12 with the X-ray crystallographic structures for this region in the p21 protein and EFtu. A model for the structure of the p21 protein in its oncogenic form

The GTP-binding p21 protein encoded by the ras-oncogene can be activated to cause malignant transformation of cells by substitution of a single amino acid at critical positions along the polypeptide chain. Substitution of any non-cyclic L-amino acid for Gly 12 in the normal protein results in a transforming protein. This substitution occurs in a hydrophobic sequence (residues 6-15) which is known to be involved in binding the phosphate moities of GTP (and GDP). We find, using conformational energy calculations, that the 6-15 segment of the normal protein (with Gly 12) adopts structures that contain a bend at residues 11 and 12 with the Gly in the D* conformation, not allowed energetically for L-amino acids. Substitution of non-cyclic L-amino acids for Gly 12 results in shifting this bend to residues 12 and 13. We show that many computed structures for the Gly 12-containing phosphate binding loop, segment 9-15, are superimposable on the corresponding segment of the recently determined X-ray crystallographic structure for residues 1-171 of the p21 protein. All such structures contain bends at residues 11 and 12 and most of these contain Gly 12 in the C* or D* conformational state. Other computed conformations for the 9-15 segment were superimposable on the structure of the corresponding 18-23 segment of EFtu, the bacterial chain elongation factor having structural similarities to the p21 protein in the phosphate-binding regions. This segment contains a Val residue where a Gly occurs in the p21 protein. As previously predicted, all of these superimposable conformations contain a bend at positions 12 and 13, not 11 and 12. If these structures that are superimposable on EFtu are introduced into the p21 protein structure, bad contacts occur between the sidechain of the residue (here Val) at position 12 and another phosphate binding loop region around position 61. These bad contacts between the two segments can be removed by changing the conformation of the 61 region in the p21 protein to the corresponding position of the homologous region in EFtu. In this new conformation, a large site becomes available for the binding of phosphate residues. In addition, such phenomena as autophosphorylation of the p21 protein by GTP can be explained with this new model structure for the activated protein which cannot be explained by the structure for the non-activated protein.     

A computational approach to explore and identify potential herbal inhibitors for the p21-activated kinase 1 (PAK1)

Abstract

The oncogenic kinase PAK1 (p21-activated kinase 1) is involved in developing many diseases including cancers, neurofibromatosis, Alzheimer's disease, diabetes (type 2), and hypertension. Thus, it is thought to be a prominent therapeutic target, and its selective inhibitors have a huge market potential. Recently, herbal PAK1 inhibitors have gained immense interest over synthetic ones mainly due to their non-toxic effects. Till date, many herbal compounds have been suggested to inhibit PAK1, but their information on selectivity, bioavailability, ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties, and molecular interactions with PAK1 has not been explored. Hence, this study was designed with computational approaches to explore and identify the best herbal PAK1-blockers showing good ADMET properties, druggable features and binding affinity with PAK1. Herbal inhibitors reported here were initially filtered with Lipinski’s rule of five (RO5). Then, molecular docking between these inhibitors and PAK1 catalytic sites was performed using AutoDock Vina and GOLD suite to determine the binding affinity and interactions. Finally, 200?ns molecular dynamics (MD) simulations on three top-ranked inhibitors including cucurbitacin I (C-I), nymphaeol A (NA), and staurosporine (SPN) were carried out. The binding free energies and interactions revealed that NA can strongly bind with the PAK1 catalytic cleft. PASS prediction and ADMET profiling supported that NA is appeared to be a more selective and safer inhibitor than C-I and SPN. These results conform to the previous experimental evidences, and therefore, NA from Okinawa propolis could be a promising inhibitor for treating PAK1-dependent illnesses.

Communicated by Ramaswamy H. Sarma     

The structure of the carboxyl terminus of the p21 protein. Structural relationship to the nucleotide-binding/transforming regions of the protein

The carboxyl-terminal region of theras oncogene-encoded p21 protein is critical to the protein's function, since membrane binding through the C-terminus is necessary for its cellular activity. X-ray crystal structures for truncated p21 proteins are available, but none of these include the C-terminal region of the protein (from residues 172–189). Using conformational energy analysis, we determined the preferred three-dimensional structures for this C-terminal octadecapeptide of the H-ras oncogene p21 protein and generated these structures onto the crystal structure of the remainder of the protein. The results indicate that, like other membrane-associated proteins, the membrane-binding C-terminus of p21 assumes a helical hairpin conformation. In several low-energy orientations, the C-terminal structure is in close proximity to other critical locales of p21. These include the central transforming region (around Gln 61) and the amino terminal transforming region (around Gly 12), indicating that extracellular signals can be transduced through the C-terminal helical hairpin to the effector regions of the protein. This finding is consistent with the results of recent genetic experiments.     

Mitochondrial ribosomal protein S36 delays cell cycle progression in association with p53 modification and p21(WAF1/CIP1) expression

Ribosomal biogenesis is correlated with cell cycle, cell proliferation, cell growth and tumorigenesis. Some oncogenes and tumor suppressors are involved in regulating the formation of mature ribosome and affecting the ribosomal biogenesis. In previous studies, the mitochondrial ribosomal protein L41 was reported to be involved in cell proliferation regulating through p21(WAF1/CIP1) and p53 pathway. In this report, we have identified a mitochondrial ribosomal protein S36 (mMRPS36), which is localized in the mitochondria, and demonstrated that overexpression of mMRPS36 in cells retards the cell proliferation and delays cell cycle progression. In addition, the mMRPS36 overexpression induces p21(WAF1/CIP1) expression, and regulates the expression and phosphorylation of p53. Our result also indicate that overexpression of mMRPS36 affects the mitochondrial function. These results suggest that mMRPS36 plays an important role in mitochondrial ribosomal biogenesis, which may cause nucleolar stress, thereby leading to cell cycle delay.     

Backbone dynamics and solution structure refinement of the 15N-labeled human oncogenic protein p13MTCP1: Comparison with X-ray data

Two related oncogenes, TCL1 and MTCP1, are overexpressed in certain T-cell prolymphocytic leukemias as a result of chromosomal rearrangements that involve the translocation of one T-cell receptor gene to either chromosome 14q32 or Xq28, respectively. The human oncoprotein p13 ^MTCP1 is coded by the MTCP1 gene and its primary sequence is highly and only homologous to that of p14 ^TCL1 , the product of TCL1. These two proteins likely represent the first members of a new family of oncogenic proteins. A previous model of the three-dimensional solution structure of p13 ^MTCP1 was determined recently using exclusively homonuclear proton two-dimensional NMR methods and, almost simultaneously, high-resolution crystal structures of p13 ^MTCP1 and p14 ^TCL1 appeared in the literature. In order to gain more insight into the details of the solution structure, we uniformly labeled p13 ^MTCP1 with nitrogen-15. The refined structure benefits from 520 additional NOEs, extracted from either ¹⁵N-edited 3D experiments or homonuclear 2D NOESY recorded at 800 MHz, and from a nearly complete set of angular restraints. Measurements of ¹⁵N spin relaxation times and heteronuclear ¹⁵N{¹H}NOEs at two magnetic field strengths provided additional insights into the dynamics of the protein backbone. On the basis of these new results, a putative binding surface for this particular class of oncogenes is discussed.     

Computed three-dimensional structures for theras-binding domain of theraf-p74 protein complexed withras-p21 and with its suppressor protein, rap-1A

The three-dimensional structures of theras-p21 protein and its protein inhibitor, rap-1A, have been computed bound to theras-binding domain, RBD (residues 55–131), of theraf-p74 protein, a critical target protein ofras-p21 in theras-induced mitogenic signal transduction pathway. The coordinates of RBD have been reconstructed from the stereoview of an X-ray crystal structure of this domain bound to rap-1A and have been subjected to energy minimization. The energy-minimized structures of bothras- p21 and rap-1A, obtained in previous studies, have been docked against RBD, using the stereo figure of the RBD-rap-1A complex, based on a six-step procedure. The final energy-minimized structure of rap-1A-RBD is identical to the X-ray crystal structure. Comparison of theras-p21- and rap-1A-RBD complexes reveals differences in the structures of effector domains ofras-p21 and rap-1a, including residues 32–47, a domain that directly interacts with RBD, 60–66, 96–110, involved in the interaction ofras-p21 withjun kinase (JNK) andjun protein, and 115–126, involved in the interaction of p21 with JNK. The structure of the RBD remained the same in both complexes with the exception of small deviations in its-2 binding loop (residues 63–71) and residues 89–91, also involved in binding to rap-1A. The results suggest that the binding of these two proteins to RBD may allow them to interact with other cellular target proteins such as JNK andjun.     

c-Abl-binding protein interacts with p21-activated kinase 2 (PAK-2) to regulate PDGF-induced membrane ruffles

p21-Activated kinases (PAKs) are serine/threonine kinases involved in multiple cellular functions including cytoskeleton regulation, proliferation and apoptosis. We performed a screen for proteins interacting with PAK-2, a ubiquitously expressed kinase involved in apoptotic signaling. Among the PAK-2 interacting proteins were different members of the Abl-binding protein family. Abl-binding proteins bound to a proline-rich region of PAK-2 located in the regulatory N terminus. Moreover, active PAK-2 phosphorylated Abl-binding proteins in vitro. Interestingly, we show that PAK-2 also interacted with c-Abl but via a different domain than with the Abl-binding proteins. PAK-2 and Abi-1 co-localized in the cytoplasm and to membrane dorsal ruffles induced by PDGF treatment. Expression of mutant PAK-2 deficient in binding to Abl-binding proteins or silencing of PAK-2 expression prevented the formation of membrane dorsal ruffles in response to PDGF. Our findings define a new class of PAK-interacting proteins, which play an important role in actin cytoskeletal reorganization.     

Activated conformations of the ras-gene-encoded p21 protein. 1. An energy-refined structure for the normal p21 protein complexed with GDP.

A complete three-dimensional structure for the ras-gene-encoded p21 protein with Gly 12 and Gln 61, bound to GDP, has been constructed in four stages using the available alpha-carbon coordinates as deposited in the Brookhaven National Laboratories Protein Data Bank. No all-atom structure has been made available despite the fact that the first crystallographic structure for the p21 protein was reported almost four years ago. In the p21 protein, if amino acid substitutions are made at any one of a number of different positions in the amino acid sequence, the protein becomes permanently activated and causes malignant transformation of normal cells or, in some cell lines, differentiation and maturation. For example, all amino acids except Gly and Pro at position 12 result in an oncogenic protein; all amino acids except Gln, Glu and Pro at position 61 likewise cause malignant transformation of cells. We have constructed our all-atom structure of the non-oncogenic protein from the x-ray structure in order to determine how oncogenic amino acid substitutions affect the three-dimensional structure of this protein. In Stage 1 we generated a poly-alanine backbone (except at Gly and Pro residues) through the alpha-carbon structure, requiring the individual Ala, Pro or Gly residues to conform to standard amino acid geometry and to form trans-planar peptide bonds. Since no alpha-carbon coordinates for residues 60-65 have been determined, these residues were modeled by generating them in the extended conformation and then subjecting them to molecular dynamics using the computer application DISCOVER and energy minimization using DISCOVER and the ECEPP (Empirical Conformational Energies for Peptides Program). In Stage 2, the positions of residues that are homologous to corresponding residues of bacterial elongation factor Tu (EF-Tu) to which p21 bears an overall 40% sequence homology, were determined from their corresponding positions in a high-resolution structure of EF-Tu. Non-homologous loops were taken from the structure generated in Stage 1 and were placed between the appropriate homologous segments so as to connect them. In Stage 3, all bad contacts that occurred in this resulting structure were removed, and the coordinates of the alpha-carbon atoms were forced to superimpose as closely as possible on the corresponding atoms of the reference (x-ray) structure. Then the side chain positions of residues of the non-homologous loop regions were modeled using a combination of molecular dynamics and energy minimization using DISCOVER and ECEPP respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

p21(Cip-1/SDI-1/WAF-1) expression via the mitogen-activated protein kinase signaling pathway in insulin-induced chondrogenic differentiation of ATDC5 cells

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin or insulin-like growth factor-I stimulation. In this study, we investigated the roles of mitogen-activated protein (MAP) kinases in insulin-induced chondrogenic differentiation of ATDC5 cells. Insulin-induced accumulation of glycosaminoglycan and expression of chondrogenic differentiation markers, type II collagen, type X collagen, and aggrecan mRNA were inhibited by the MEK1/2 inhibitor (U0126) and the p38 MAP kinase inhibitor (SB203580). Conversely, the JNK inhibitor (SP600125) enhanced the synthesis of glycosaminoglycan and expression of chondrogenic differentiation markers. Insulin-induced phosphorylation of ERK1/2 and JNK but not that of p38 MAP kinase. We have previously clarified that the induction of the cyclin-dependent kinase inhibitor, p21(Cip-1/SDI-1/WAF-1), is essential for chondrogenic differentiation of ATDC5 cells. To assess the relationship between the induction of p21 and MAP kinase activity, we investigated the effect of these inhibitors on insulin-induced p21 expression in ATDC5 cells. Insulin-induced accumulation of p21 mRNA and protein was inhibited by the addition of U0126 and SB203580. In contrast, SP600125 enhanced it. Inhibitory effects of U0126 or stimulatory effects of SP600125 on insulin-induced chondrogenic differentiation were observed when these inhibitors exist in the early phase of differentiation, suggesting that MEK/ERK and JNK act on early phase differentiation. SB202580, however, is necessary not only for early phase but also for late phase differentiation, indicating that p38 MAP kinase stimulates differentiation by acting during the entire period of cultivation. These results for the first time demonstrate that up-regulation of p21 expression by ERK1/2 and p38 MAP kinase is required for chondrogenesis, and that JNK acts as a suppressor of chondrogenesis by down-regulating p21 expression.     

ARF-dependent regulation of ATM and p53 associated KZNF (Apak) protein activity in response to oncogenic stress

The KRAB-type zinc-finger protein Apak (ATM and p53 associated KZNF protein) specifically suppresses p53-mediated apoptosis. Upon DNA damage, Apak is phosphorylated and inhibited by ATM kinase, resulting in p53 activation. However, how Apak is regulated in response to oncogenic stress remains unknown. Here we show that upon oncogene activation, Apak is inhibited in the tumor suppressor ARF-dependent but ATM-independent manner. Oncogene-induced ARF protein directly interacts with Apak and competes with p53 to bind to Apak, resulting in Apak dissociation from p53. Thus, Apak is differentially regulated in the ARF and ATM-dependent manner in response to oncogenic stress and DNA damage, respectively.

Structured summary

MINT-7989670: p53 (uniprotkb:P04637) binds (MI:0407) to APAK (uniprotkb:Q8TAQ5) by pull down (MI:0096)MINT-7989812: HDM2 (uniprotkb:Q00987) physically interacts (MI:0915) with ARF (uniprotkb:Q8N726-1) by anti bait coimmunoprecipitation (MI:0006)MINT-7989603, MINT-7989626: APAK (uniprotkb:Q8TAQ5) physically interacts (MI:0915) with ARF (uniprotkb:Q8N726-1) by anti bait coimmunoprecipitation (MI:0006)MINT-7989653: ARF (uniprotkb:Q8N726-1) binds (MI:0407) to APAK (uniprotkb:Q8TAQ5) by pull down (MI:0096)MINT-7989686, MINT-7989705, MINT-7989747:APAK (uniprotkb:Q8TAQ5) physically interacts (MI:0915) with ARF (uniprotkb:Q8N726-1) by anti tag coimmunoprecipitation (MI:0007)MINT-7989724: APAK (uniprotkb:Q8TAQ5) physically interacts (MI:0914) with ARF (uniprotkb:Q8N726-1) and p53 (uniprotkb:P04637) by anti tag coimmunoprecipitation (MI:0007)MINT-7989635: ARF (uniprotkb:Q8N726-1) and APAK (uniprotkb:Q8TAQ5) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7989584, MINT-7989773: APAK (uniprotkb:Q8TAQ5) physically interacts (MI:0915) with p53 (uniprotkb:P04637) by anti tag coimmunoprecipitation (MI:0007)