Increased risk of kidney failure in patients with genetic kidney disorders.

BACKGROUNDIt is unknown whether the risk of kidney disease progression and failure differs between patients with and without genetic kidney disorders.METHODSThree cohorts were evaluated: the prospective Cure Glomerulonephropathy Network (CureGN) and 2 retrospective cohorts from Columbia University, including 5,727 adults and children with kidney disease from any etiology who underwent whole-genome or exome sequencing. The effects of monogenic kidney disorders and APOL1 kidney-risk genotypes on the risk of kidney failure, estimated glomerular filtration rate (eGFR) decline, and disease remission rates were evaluated along with diagnostic yields and the impact of American College of Medical Genetics secondary findings (ACMG SFs).RESULTSMonogenic kidney disorders were identified in 371 patients (6.5%), high-risk APOL1 genotypes in 318 (5.5%), and ACMG SFs in 100 (5.2%). Family history of kidney disease was the strongest predictor of monogenic disorders. After adjustment for traditional risk factors, monogenic kidney disorders were associated with an increased risk of kidney failure (hazard ratio [HR] = 1.72), higher rate of eGFR decline (-3.06 vs. 0.25 mL/min/1.73 m2/year), and lower risk of complete remission (odds ratioNot achieving CR = 5.25). High-risk APOL1 genotypes were associated with an increased risk of kidney failure (HR = 1.67) and faster eGFR decline (-2.28 vs. 0.25 mL/min/1.73 m2), replicating prior findings. ACMG SFs were not associated with personal or family history of associated diseases, but were predicted to impact care in 70% of cases.CONCLUSIONSMonogenic kidney disorders were associated with an increased risk of kidney failure, faster eGFR decline, and lower rates of complete remission, suggesting opportunities for early identification and intervention based on molecular diagnosis.TRIAL REGISTRATIONNA.FUNDINGNational Institute of Diabetes and Digestive and Kidney Diseases grants U24DK100845 (formerly UM1DK100845), U01DK100846 (formerly UM1DK100846), U01DK100876 (formerly UM1DK100876), U01DK100866 (formerly UM1DK100866), U01DK100867 (formerly UM1DK100867), U24DK100845, DK081943, RC2DK116690, 2U01DK100876, 1R01DK136765, 5R01DK082753, and RC2-DK122397; NephCure Kidney International; Department of Defense Research Awards PR201425, W81XWH-16-1-0451, and W81XWH-22-1-0966; National Center for Advancing Translational Sciences grant UL1TR001873; National Library of Medicine grant R01LM013061; National Human Genome Research Institute grant 2U01HG008680.

The Clinical Utility of Genetic Testing in the Diagnosis and Management of Adults with Chronic Kidney Disease.

SIGNIFICANCE STATEMENT: Accurate diagnosis of a patient's underlying cause of CKD can influence management and ultimately overall health. The single-arm, interventional, prospective Renasight Clinical Application, Review, and Evaluation study assessed the utility of genetic testing with a 385 gene kidney disease panel on the diagnosis and management of 1623 patients with CKD. Among 20.8% of patients who had positive genetic findings, half resulted in a new or reclassified diagnosis. In addition, a change in management because of genetic testing was reported for 90.7% of patients with positive findings, including treatment changes in 32.9%. These findings demonstrate that genetic testing has a significant effect on both CKD diagnosis and management. BACKGROUND: Genetic testing in CKD has recently been shown to have diagnostic utility with many predicted implications for clinical management, but its effect on management has not been prospectively evaluated. METHODS: Renasight Clinical Application, Review, and Evaluation RenaCARE (ClinicalTrials.gov NCT05846113 ) is a single-arm, interventional, prospective, multicenter study that evaluated the utility of genetic testing with a broad, 385 gene panel (the Renasight TM test) on the diagnosis and management of adult patients with CKD recruited from 31 US-based community and academic medical centers. Patient medical history and clinical CKD diagnosis were collected at enrollment. Physician responses to questionnaires regarding patient disease categorization and management were collected before genetic testing and 1 month after the return of test results. Changes in CKD diagnosis and management after genetic testing were assessed. RESULTS: Of 1623 patients with CKD in 13 predefined clinical disease categories (ages, 18-96; median, 55 years), 20.8% ( n =338) had positive genetic findings spanning 54 genes. Positive genetic findings provided a new diagnosis or reclassified a prior diagnosis in 48.8% of those patients. Physicians reported that genetic results altered the management of 90.7% of patients with a positive genetic finding, including changes in treatment plan, which were reported in 32.9% of these patients. CONCLUSIONS: Genetic testing with a CKD-focused 385 gene panel substantially refined clinical diagnoses and had widespread implications for clinical management, including appropriate treatment strategies. These data support the utility of broader integration of panels of genetic tests into the clinical care paradigm for patients with CKD. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: ClinicalTrials.gov, NCT05846113 .

Membrane-type 1 Matrix Metalloproteinase Modulates Tissue Homeostasis by a Non-proteolytic Mechanism.

Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane proteinase with a short cytoplasmic tail, is a major effector of extracellular matrix remodeling. Genetic silencing of MT1-MMP in mouse (Mmp14 -/- ) and man causes dwarfism, osteopenia, arthritis, and lipodystrophy, abnormalities ascribed to defective collagen turnover. We have previously shown non-proteolytic functions of MT1-MMP mediated by its cytoplasmic tail, where the unique tyrosine (Y573) controls intracellular signaling. The Y573D mutation blocks TIMP-2/MT1-MMP-induced Erk1/2 and Akt signaling without affecting proteolytic activity. Here, we report that a mouse with the MT1-MMP Y573D mutation (Mmp14 Y573D/Y573D ) shows abnormalities similar to but also different from those of Mmp14 -/- mice. Skeletal stem cells (SSC) of Mmp14 Y573D/Y573D mice show defective differentiation consistent with the mouse phenotype, which is rescued by wild-type SSC transplant. These results provide the first in vivo demonstration that MT1-MMP modulates bone, cartilage, and fat homeostasis by controlling SSC differentiation through a mechanism independent of proteolysis.

Abaloparatide at the Same Dose Has the Same Effects on Bone as PTH (1-34) in Mice.

Abaloparatide, a novel analog of parathyroid hormone-related protein (PTHrP 1-34), became in 2017 the second osteoanabolic therapy for the treatment of osteoporosis. This study aims to compare the effects of PTH (1-34), PTHrP (1-36), and abaloparatide on bone remodeling in male mice. Intermittent daily subcutaneous injections of 80 µg/kg/d were administered to 4-month-old C57Bl/6J male mice for 6 weeks. During treatment, mice were followed by DXA-Piximus to assess changes in bone mineral density (BMD) in the whole body, femur, and tibia. At either 4 or 18 hours after the final injection, femurs were harvested for µCT analyses and histomorphometry, sera were assayed for bone turnover marker levels, and tibias were separated into cortical, trabecular, and bone marrow fractions for gene expression analyses. Our results showed that, compared with PTH (1-34), abaloparatide resulted in a similar increase in BMD at all sites, whereas no changes were found with PTHrP (1-36). With both PTH (1-34) and abaloparatide, µCT and histomorphometry analyses revealed similar increases in bone volume associated with an increased trabecular thickness, in bone formation rate as shown by P1NP serum level and in vivo double labeling, and in bone resorption as shown by CTX levels and osteoclast number. Gene expression analyses of trabecular and cortical bone showed that PTH (1-34) and abaloparatide led to different actions in osteoblast differentiation and activity, with increased Runx2, Col1A1, Alpl, Bsp, Ocn, Sost, Rankl/Opg, and c-fos at different time points. Abaloparatide seems to generate a faster response on osteoblastic gene expression than PTH (1-34). Taken together, abaloparatide at the same dose is as effective as PTH (1-34) as an osteoanabolic, with an increase in bone formation but also an increase in bone resorption in male mice. © 2019 American Society for Bone and Mineral Research.

Serine-threonine protein phosphatases: Lost in translation.

Protein synthesis is one of the most complex and energy-consuming processes in eukaryotic cells and therefore is tightly regulated. One of the main mechanisms of translational control is post-translational modifications of the components of translational apparatus. Phosphorylation status of translation factors depends on the balanced action of kinases and phosphatases. While many kinase-dependent events are well defined, phosphatases that counteract phosphorylation are rarely determined. This mini-review focuses on the regulation of activity of translational initiation factors by serine/threonine phosphatases.

Parathyroid hormone(1-34) and its analogs differentially modulate osteoblastic Rankl expression via PKA/SIK2/SIK3 and PP1/PP2A-CRTC3 signaling.

Osteoporosis can result from the loss of sex hormones and/or aging. Abaloparatide (ABL), an analog of parathyroid hormone-related protein (PTHrP(1-36)), is the second osteoanabolic therapy approved by the United States Food and Drug Administration after teriparatide (PTH(1-34)). All three peptides bind PTH/PTHrP receptor type 1 (PTHR1), but the effects of PTHrP(1-36) or ABL in the osteoblast remain unclear. We show that, in primary calvarial osteoblasts, PTH(1-34) promotes a more robust cAMP response than PTHrP(1-36) and ABL and causes a greater activation of protein kinase A (PKA) and cAMP response element-binding protein (CREB). All three peptides similarly inhibited sclerostin (Sost). Interestingly, the three peptides differentially modulated two other PKA target genes, c-Fos and receptor activator of NF-κB ligand (Rankl), and the latter both in vitro and in vivo Knockdown of salt-inducible kinases (SIKs) 2 and 3 and CREB-regulated transcription coactivator 3 (CRTC3), indicated that all three are part of the pathway that regulates osteoblastic Rankl expression. We also show that the peptides differentially regulate the nuclear localization of CRTC2 and CRTC3, and that this correlates with PKA activation. Moreover, inhibition of protein phosphatases 1 and 2A (PP1/PP2A) activity revealed that they play a major role in both PTH-induced Rankl expression and the effects of PTH(1-34) on CRTC3 localization. In summary, in the osteoblast, the effects of PTH(1-34), PTHrP(1-36), and ABL on Rankl are mediated by differential stimulation of cAMP/PKA signaling and by their downstream effects on SIK2 and -3, PP1/PP2A, and CRTC3.

mTOR-mediated inactivation of 4E-BP1, an inhibitor of translation, precedes cartilage degeneration in rat osteoarthritic knees.

Proper control of protein synthesis is vital for tissue homeostasis and its deregulation is characteristic of many disorders including osteoarthritis (OA). The objectives of this work were to analyze and correlate changes in activity of the translation apparatus associated with cartilage degeneration in an animal model of OA. Osteoarthritis was induced surgically in rats by anterior cruciate ligament transection (ACLT). Using a modified Mankin scoring system and analysis of protein expression we demonstrated, that mechanistic target of rapamycin complex 1 (mTORC1)-mediated 4E-BP1 phosphorylation was detected significantly earlier than other mTORC1-mediated modifications, such as p70S6K and ULK1 phosphorylation. 4E-BP1 is an inhibitor of cap-dependent translation those functions are inhibited by mTORC1 mediated phosphorylation. This signaling event not only preceded prominent signs of cartilage degeneration but also the increase in global protein synthesis rate. These results suggest that abnormal mTORC1 activity is one of the primary dysregulations observed in OA cartilage. Importantly, it is distributed disproportionately between targets, with 4E-BP1 being phosphorylated earlier than other downstream targets. Thus, our work provides new insights into the sequence of molecular events leading to cartilage destruction in OA and identifies translational control as an important regulatory hub involved in initiating OA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2728-2735, 2018.

Phosphoproteomics of Fibroblast Growth Factor 1 (FGF1) Signaling in Chondrocytes: Identifying the Signature of Inhibitory Response.

Fibroblast growth factor (FGF) signaling is vital for many biological processes, beginning with development. The importance of FGF signaling for skeleton formation was first discovered by the analysis of genetic FGFR mutations which cause several bone morphogenetic disorders, including achondroplasia, the most common form of human dwarfism. The formation of the long bones is mediated through proliferation and differentiation of highly specialized cells - chondrocytes.Chondrocytes respond to FGF with growth inhibition, a unique response which differs from the proliferative response of the majority of cell types; however, its molecular determinants are still unclear. Quantitative phosphoproteomic analysis was utilized to catalogue the proteins whose phosphorylation status is changed upon FGF1 treatment. The generated dataset consists of 756 proteins. We could localize the divergence between proliferative (canonical) and inhibitory (chondrocyte specific) FGF transduction pathways immediately upstream of AKT kinase. Gene Ontology (GO) analysis of the FGF1 regulated peptides revealed that many of the identified phosphorylated proteins are assigned to negative regulation clusters, in accordance with the observed inhibitory growth response. This is the first time a comprehensive subset of proteins involved in FGF inhibitory response is defined. We were able to identify a number of targets and specifically discover glycogen synthase kinase3ß (GSK3ß) as a novel key mediator of FGF inhibitory response in chondrocytes.

Increased Activity of the Chondrocyte Translational Apparatus Accompanies Osteoarthritic Changes in Human and Rodent Knee Cartilage.

OBJECTIVE: Degeneration of articular cartilage is central to the pathology of osteoarthritis (OA). However, the molecular mechanisms leading to these irreversible changes are still poorly understood. This study was undertaken to investigate how changes in the chondrocyte translational apparatus may contribute to the development and progression of knee OA. METHODS: Articular cartilage from the knees of normal healthy subjects and patients with OA was used to analyze the activity of different components of the translational machinery. Chondrocytes isolated from lesional and nonlesional areas of the human OA cartilage were used to estimate the relative rate of protein synthesis by metabolic labeling. Experimental OA was induced by transection of the anterior cruciate ligament of rats to investigate changes in the translational apparatus associated with OA. The role of interleukin-1ß (IL-1ß) signaling was assessed in vitro using rat articular chondrocytes. In human or rodent knee cartilage, messenger RNA expression was analyzed by quantitative polymerase chain reaction, and protein levels were determined by immunohistochemistry and Western blotting. RESULTS: Several novel traits of OA chondrocytes were identified, including up-regulation of the serine/threonine kinases Akt-2 and Akt-3 at the posttranscriptional level and an increased rate of total protein synthesis, likely attributable to inactivation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1), a known repressor of cap-dependent translation. Inactivation of 4E-BP1 was dependent on the activity of mechanistic target of rapamycin and was crucial for the up-regulation of protein synthesis in general and expression of matrix metalloproteinase 13 and ADAMTS-5 in particular. In addition, treatment of articular chondrocytes with IL-1ß led to inactivation of 4E-BP1 and up-regulation of protein synthesis. CONCLUSION: Precise control of protein synthesis is vital for cartilage homeostasis, and its dysregulation contributes to the molecular pathology of OA. The results of this study therefore identify a novel set of potential therapeutic targets to ameliorate the effects of knee OA.

Cell type-specific control of protein synthesis and proliferation by FGF-dependent signaling to the translation repressor 4E-BP.

Regulation of protein synthesis plays a vital role in posttranscriptional modulation of gene expression. Translational control most commonly targets the initiation of protein synthesis: loading 40S ribosome complexes onto mRNA and AUG start codon recognition. This step is initiated by eukaryotic initiation factor 4E (eIF4E) (the m7GTP cap-binding protein), whose binding to eIF4G (a scaffolding subunit) and eIF4A (an ATP-dependent RNA helicase) leads to assembly of active eIF4F complex. The ability of eIF4E to recognize the cap is prevented by its binding to eIF4E binding protein (4E-BP), which thereby inhibits cap-dependent translation by sequestering eIF4E. The 4E-BP activity is, in turn, inhibited by mTORC1 [mTOR (the mechanistic target of rapamycin) complex 1] mediated phosphorylation. Here, we define a previously unidentified mechanism of mTOR-independent 4E-BP1 regulation that is used by chondrocytes upon FGF signaling. Chondrocytes are responsible for the formation of the skeleton long bones. Unlike the majority of cell types where FGF signaling triggers proliferation, chondrocytes respond to FGF with inhibition. We establish that FGF specifically suppresses protein synthesis in chondrocytes, but not in any other cells of mesenchymal origin. Furthermore, 4E-BP1 repressor activity is necessary not only for suppression of protein synthesis, but also for FGF-induced cell-cycle arrest. Importantly, FGF-induced changes in the 4E-BP1 activity observed in cell culture are likewise detected in vivo and reflect the action of FGF signaling on downstream targets during bone development. Thus, our findings demonstrate that FGF signaling differentially impacts protein synthesis through either stimulation or repression, in a cell-type-dependent manner, with 4E-BP1 being a key player.

The B55α regulatory subunit of protein phosphatase 2A mediates fibroblast growth factor-induced p107 dephosphorylation and growth arrest in chondrocytes.

Fibroblast growth factor (FGF)-induced growth arrest of chondrocytes is a unique cell type-specific response which contrasts with the proliferative response of most cell types and underlies several genetic skeletal disorders caused by activating FGF receptor (FGFR) mutations. We have shown that one of the earliest key events in FGF-induced growth arrest is dephosphorylation of the retinoblastoma protein (Rb) family member p107 by protein phosphatase 2A (PP2A), a ubiquitously expressed multisubunit phosphatase. In this report, we show that the PP2A-B55α holoenzyme (PP2A containing the B55α subunit) is responsible for this phenomenon. Only the B55α (55-kDa regulatory subunit, alpha isoform) regulatory subunit of PP2A was able to bind p107, and this interaction was induced by FGF in chondrocytes but not in other cell types. Small interfering RNA (siRNA)-mediated knockdown of B55α prevented p107 dephosphorylation and FGF-induced growth arrest of RCS (rat chondrosarcoma) chondrocytes. Importantly, the B55α subunit bound with higher affinity to dephosphorylated p107. Since the p107 region interacting with B55α is also the site of cyclin-dependent kinase (CDK) binding, B55α association may also prevent p107 phosphorylation by CDKs. FGF treatment induces dephosphorylation of the B55α subunit itself on several serine residues that drastically increases the affinity of B55α for the PP2A A/C dimer and p107. Together these observations suggest a novel mechanism of p107 dephosphorylation mediated by activation of PP2A through B55α dephosphorylation. This mechanism might be a general signal transduction pathway used by PP2A to initiate cell cycle arrest when required by external signals.

PP1 and PP2A phosphatases--cooperating partners in modulating retinoblastoma protein activation.

The retinoblastoma/pocket protein family is one of the master regulators of the eukaryotic cell cycle. It includes the retinoblastoma protein (Rb) and the related p107 and p130 proteins. The importance of the Rb pathway for homeostasis and tumour suppression is evident from the fact that inactivating mutations in Rb are frequently associated with many cancers. Rbs regulate the cell cycle by controlling the activity of the E2F family of transcription factors. The activity of Rb proteins themselves is modulated by their phosphorylation status at several Ser/Thr residues: phosphorylation by cyclin-dependent kinases inactivates Rb proteins and positively influences the transcription of genes necessary for cell cycle progression. Although the mechanisms of cyclin-dependent kinase-mediated inactivation of Rb proteins are understood in great detail, our knowledge of the process that counteracts Rb phosphorylation is still quite limited. The present review focuses on the Ser/Thr phosphatases that are responsible for the dephosphorylation and thus activation of Rb proteins. Two major scenarios are considered: (a) when pocket proteins are dephosphorylated during regular cell cycle progression and (b) when rapid dephosphorylation is dictated by external stress or growth inhibitory conditions, such as oxidative stress, UV radiation or other DNA-damaging stimuli, and cell differentiation factors. It transpires that protein phosphatase 1 and protein phosphatase 2A can efficiently modulate pocket protein activity in a highly context-dependent manner and both are tightly regulated by the presence of different regulatory subunits or interacting proteins.

Overexpression of cyclin E/CDK2 complexes overcomes FGF-induced cell cycle arrest in the presence of hypophosphorylated Rb proteins.

FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent. To examine the role of cyclin E/CDK2 complexes in mediating FGF-induced growth arrest, this kinase was overexpressed in RCS cells. FGF-induced dephosphorylation of either p107 or p130 was not prevented by overexpressing cyclin E/CDK2 complexes. Unexpectedly, however, FGF-treated cells exhibited sustained proliferation even in the presence of hypophosphorylated p107 and p130. Both pocket proteins were able to form repressive complexes with E2F4 and E2F5 but these repressors were not translocated into the nucleus and therefore were unable to occupy their respective target DNA sites. Overexpressed cyclin E/CDK2 molecules were stably associated with p107 and p130 in FGF-treated cells in the context of E2F repressive complexes. Taken together, our data suggest a novel mechanism by which cyclin E/CDK2 complexes can promote cell cycle progression in the presence of dephosphorylated Rb proteins and provide a novel insight into the key Retinoblastoma/E2F/cyclin E pathway. Our data also highlight the importance of E2F4/p130 complexes for FGF-mediated growth arrest in chondrocytes.

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G2arrest in RCS chondrocytes.

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G1 phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G2 phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G1 arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. the inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G2 arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and AtM/ATR kinase are known to play essential roles in the G2 checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G2 arrest. Additionally our results indicate that the transient G2 arrest is induced by FGF in RCS cell through mechanisms that are independent of the G1 arrest, and that the G2 block is not strictly required for the sustained G1 arrest but may provide a pausing mechanism that allows the FGF response to be fully established.

Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing.

The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNA(i)(Met) attachment to 40S ribosomal subunits, scanning, start codon selection and subunit joining. Its bacterial ortholog IF1 consists of an oligonucleotide/oligosaccharide-binding (OB) domain, whereas eIF1A additionally contains a helical subdomain, N-terminal tail (NTT) and C-terminal tail (CTT). The NTT and CTT both enhance ribosomal recruitment of eIF2/GTP/Met-tRNA(i)(Met), but have opposite effects on the stringency of start codon selection: the CTT increases, whereas the NTT decreases it. Here, we determined the position of eIF1A on the 40S subunit by directed hydroxyl radical cleavage. eIF1A's OB domain binds in the A site, similar to IF1, whereas the helical subdomain contacts the head, forming a bridge over the mRNA channel. The NTT and CTT both thread under Met-tRNA(i)(Met) reaching into the P-site. The NTT threads closer to the mRNA channel. In the proposed model, the NTT does not clash with either mRNA or Met-tRNA(i)(Met), consistent with its suggested role in promoting the 'closed' conformation of ribosomal complexes upon start codon recognition. In contrast, eIF1A-CTT appears to interfere with the P-site tRNA-head interaction in the 'closed' complex and is likely ejected from the P-site upon start codon recognition.

PP2A-mediated dephosphorylation of p107 plays a critical role in chondrocyte cell cycle arrest by FGF.

FGF signaling inhibits chondrocyte proliferation, a cell type-specific response that is the basis for several genetic skeletal disorders caused by activating FGFR mutations. This phenomenon requires the function of the p107 and p130 members of the Rb protein family, and p107 dephosphorylation is one of the earliest distinguishing events in FGF-induced growth arrest. To determine whether p107 dephoshorylation played a critical role in the chondrocyte response to FGF, we sought to counteract this process by overexpressing in RCS chondrocytes the cyclin D1/cdk4 kinase complex. CyclinD/cdk4-expressing RCS cells became resistant to FGF-induced p107 dephosphorylation and growth arrest, and maintained significantly high levels of cyclin E/cdk2 activity and of phosphorylated p130 at later times of FGF treatment. We explored the involvement of a phosphatase in p107 dephosphorylation. Expression of the SV40 small T-Ag, which inhibits the activity of the PP2A phosphatase, or knockdown of the expression of the PP2A catalytic subunit by RNA interference prevented p107 dephosphorylation and FGF-induced growth arrest of RCS cells. Furthermore, an association between p107 and PP2A was induced by FGF treatment. Our data show that p107 dephosphorylation is a key event in FGF-induced cell cycle arrest and indicate that in chondrocytes FGF activates the PP2A phosphatase to promote p107 dephosphorylation.

Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes.

The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions (+)11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions (-)3/(-)4 to rpS5(S7p) and h23b, (-)6/(-)7 to rpS14(S11p), and (-)8-(-)11 to the 3'-terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions (+)7/(+)8 to the central region of h28, (+)4/(+)5 to rpS15(S19p), and (-)6 and (-)7/(-)10 to eukaryote-specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2alpha (eIF2alpha) contacts position (-)3, and now report that eIF3 interacts with positions (-)8-(-)17, forming an extension of the mRNA-binding channel that likely contributes to unique aspects of eukaryotic initiation.

In vitro reconstitution and biochemical characterization of translation initiation by internal ribosomal entry.

The internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV) and related viruses promote initiation of translation by a noncanonical end-independent mechanism. To characterize this mechanism at the molecular level, we have developed biochemical approaches to reconstitute the process in vitro from individual purified components of the translation apparatus, developed methods to characterize steps in this process so that the functions of individual proteins can be characterized, and adapted assays such as primer extension inhibition ("toe printing") to monitor accurate assembly on the IRES of ribosomal 48S and 80S complexes. In vitro reconstitution of 48S complex formation offers an approach for the functional identification of IRES trans-acting factors (ITAFs) that are required for initiation in addition to canonical initiation factors and revealed that despite being related, different EMCV-like IRESs nevertheless have distinct ITAF requirements. Toe printing revealed that a common feature of initiation on EMCV-like IRESs is the stable binding of an eIF4G/eIF4A complex to them near the initiation codon, where it can locally unwind RNA to facilitate ribosomal attachment. The same toe printing assay indicated that binding of ITAFs to these IRESs enhances binding of these two canonical initiation factors. We also describe protocols for chemical and enzymatic footprinting to determine the interactions of trans-acting factors with the IRES at nucleotide resolution and for directed hydroxyl radical probing to determine their orientation on the IRES.

Specific functional interactions of nucleotides at key -3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex.

Eukaryotic initiation factor (eIF) 1 maintains the fidelity of initiation codon selection and enables mammalian 43S preinitiation complexes to discriminate against AUG codons with a context that deviates from the optimum sequence GCC(A/G)CCAUGG, in which the purines at (-)3 and (+)4 positions are most important. We hypothesize that eIF1 acts by antagonizing conformational changes that occur in ribosomal complexes upon codon-anticodon base-pairing during 48S initiation complex formation, and that the role of (-)3 and (+)4 context nucleotides is to stabilize these changes by interacting with components of this complex. Here we report that U and G at (+)4 both UV-cross-linked to ribosomal protein (rp) S15 in 48S complexes. However, whereas U cross-linked strongly to C(1696) and less well to AA(1818-1819) in helix 44 of 18S rRNA, G cross-linked exclusively to AA(1818-1819). U at (-)3 cross-linked to rpS5 and eIF2alpha, whereas G cross-linked only to eIF2alpha. Results of UV cross-linking experiments and of assays of 48S complex formation done using alpha-subunit-deficient eIF2 indicate that eIF2alpha's interaction with the (-)3 purine is responsible for recognition of the (-)3 context position by 43S complexes and suggest that the (+)4 purine/AA(1818-1819) interaction might be responsible for recognizing the (+)4 position.

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association.

The multisubunit eukaryotic initiation factor (eIF) 3 plays various roles in translation initiation that all involve interaction with 40S ribosomal subunits. eIF3 can be purified in two forms: with or without the loosely associated eIF3j subunit (eIF3j+ and eIF3j-, respectively). Although unlike eIF3j+, eIF3j- does not bind 40S subunits stably enough to withstand sucrose density gradient centrifugation, we found that in addition to the known stabilization of the eIF3/40S subunit interaction by the eIF2*GTP*Met-tRNA(i)Met ternary complex, eIF3j-/40S subunit complexes were also stabilized by single-stranded RNA or DNA cofactors that were at least 25 nt long and could be flanked by stable hairpins. Of all homopolymers, oligo(rU), oligo(dT), and oligo(dC) stimulated the eIF3/40S subunit interaction, whereas oligo(rA), oligo(rG), oligo(rC), oligo(dA), and oligo(dG) did not. Oligo(U) or oligo(dT) sequences interspersed by other bases also promoted this interaction. The ability of oligonucleotides to stimulate eIF3/40S subunit association correlated with their ability to bind to the 40S subunit, most likely to its mRNA-binding cleft. Although eIF3j+ could bind directly to 40S subunits, neither eIF3j- nor eIF3j+ alone was able to dissociate 80S ribosomes or protect 40S and 60S subunits from reassociation. Significantly, the dissociation/anti-association activities of both forms of eIF3 became apparent in the presence of either eIF2-ternary complexes or any oligonucleotide cofactor that promoted eIF3/40S subunit interaction. Ribosomal dissociation and anti-association activities of eIF3 were strongly enhanced by eIF1. The potential biological role of stimulation of eIF3/40S subunit interaction by an RNA cofactor in the absence of eIF2-ternary complex is discussed.