Phosphorylation of the RB C-terminus regulates condensin II release from chromatin.

The retinoblastoma tumor suppressor protein (RB) plays an important role in biological processes such as cell cycle control, DNA damage repair, epigenetic regulation, and genome stability. The canonical model of RB regulation is that cyclin-CDKs phosphorylate and render RB inactive in late G1/S, promoting entry into S phase. Recently, monophosphorylated RB species were described to have distinct cell-cycle-independent functions, suggesting that a phosphorylation code dictates diversity of RB function. However, a biologically relevant, functional role of RB phosphorylation at non-CDK sites has remained elusive. Here, we investigated S838/T841 dual phosphorylation, its upstream stimulus, and downstream functional output. We found that mimicking T-cell receptor activation in Jurkat leukemia cells induced sequential activation of downstream kinases including p38 MAPK and RB S838/T841 phosphorylation. This signaling pathway disrupts RB and condensin II interaction with chromatin. Using cells expressing a WT or S838A/T841A mutant RB fragment, we present evidence that deficiency for this phosphorylation event prevents condensin II release from chromatin.

ortho-Methoxyphenols as Convenient Oxidative Bioconjugation Reagents with Application to Site-Selective Heterobifunctional Cross-Linkers.

The synthesis of complex protein-based bioconjugates has been facilitated greatly by recent developments in chemoselective methods for biomolecular modification. The oxidative coupling of o-aminophenols or catechols with aniline functional groups is chemoselective, mild, and rapid; however, the oxidatively sensitive nature of the electron-rich aromatics and the paucity of commercial sources pose some obstacles to the general use of these reactive strategies. Herein, we identify o-methoxyphenols as air-stable, commercially available derivatives that undergo efficient oxidative couplings with anilines in the presence of periodate as oxidant. Mechanistic considerations informed the development of a preoxidation protocol that can greatly reduce the amount of periodate necessary for effective coupling. The stability and versatility of these reagents was demonstrated through the synthesis of complex protein-protein bioconjugates using a site-selective heterobifunctional cross-linker comprising both o-methoxyphenol and 2-pyridinecarboxaldehyde moieties. This compound was used to link epidermal growth factor to genome-free MS2 viral capsids, affording nanoscale delivery vectors that can target a variety of cancer cell types.

One-step site-specific modification of native proteins with 2-pyridinecarboxyaldehydes.

The chemical modification of proteins is an enabling technology for many scientific fields, including chemical biology, biophysics, bioengineering and materials science. These methods allow the attachment of strategically selected detection probes, polymers, drug molecules and analysis platforms. However, organic reactions that can proceed under conditions mild enough to maintain biomolecular function are limited. Even more rare are chemical strategies that can target a single site, leading to products with uniform properties and optimal function. We present a versatile method for the selective modification of protein N termini that does not require any genetic engineering of the protein target. This reaction is demonstrated for 12 different proteins, including the soluble domain of the human estrogen receptor. The function of this protein was confirmed through the binding of a fluorescent estrogen mimic, and the modified protein was explored as a prototype for the detection of endocrine-disrupting chemicals in water.

Capture and Recycling of Sortase†A through Site-Specific Labeling with Lithocholic Acid.

Enzyme-mediated protein modification often requires large amounts of biocatalyst, adding significant costs to the process and limiting industrial applications. Herein, we demonstrate a scalable and straightforward strategy for the efficient capture and recycling of enzymes using a small-molecule affinity tag. A proline variant of an evolved sortase A (SrtA 7M) was N-terminally labeled with lithocholic acid (LA)-an inexpensive bile acid that exhibits strong binding to ß-cyclodextrin (ßCD). Capture and recycling of the LA-Pro-SrtA 7M conjugate was achieved using ßCD-modified sepharose resin. The LA-Pro-SrtA 7M conjugate retained full enzymatic activity, even after multiple rounds of recycling.

Netrin signaling mediates survival of dormant epithelial ovarian cancer cells.

Dormancy in cancer is a clinical state in which residual disease remains undetectable for a prolonged duration. At a cellular level, rare cancer cells cease proliferation and survive chemotherapy and disseminate disease. We created a suspension culture model of high-grade serous ovarian cancer (HGSOC) dormancy and devised a novel CRISPR screening approach to identify survival genes in this context. In combination with RNA-seq, we discovered the Netrin signaling pathway as critical to dormant HGSOC cell survival. We demonstrate that Netrin-1, -3, and its receptors are essential for low level ERK activation to promote survival, and that Netrin activation of ERK is unable to induce proliferation. Deletion of all UNC5 family receptors blocks Netrin signaling in HGSOC cells and compromises viability during the dormancy step of dissemination in xenograft assays. Furthermore, we demonstrate that Netrin-1 and -3 overexpression in HGSOC correlates with poor outcome. Specifically, our experiments reveal that Netrin overexpression elevates cell survival in dormant culture conditions and contributes to greater spread of disease in a xenograft model of abdominal dissemination. This study highlights Netrin signaling as a key mediator HGSOC cancer cell dormancy and metastasis.

High-grade serous ovarian cancer (or HGSOC for short) is the fifth leading cause of cancer-related deaths in women. It is generally diagnosed at an advanced stage of disease when the cancer has already spread to other parts of the body. Surgical removal of tumors and subsequent treatment with chemotherapy often reduces the signs and symptoms of the disease for a time but some cancer cells tend to survive so that patients eventually relapse. The HGSOC cells typically spread from the ovaries by moving through the liquid surrounding organs in the abdomen. The cells clump together and enter an inactive state known as dormancy that allows them to survive chemotherapy and low-nutrient conditions. Understanding how to develop new drug therapies that target dormant cancer cells is thought to be an important step in prolonging the life of HGSOC patients. Cancer cells are hardwired to multiply and grow, so Perampalam et al. reasoned that becoming dormant poses challenges for HGSOC cells, which may create unique vulnerabilities not shared by proliferating cancer cells. To find out more, the researchers used HGSOC cells that had been isolated from patients and grown in the laboratory. The team used a gene editing technique to screen HGSOC cells for genes required by the cells to survive when they are dormant. The experiments found that genes involved in a cell signaling pathway, known as Netrin signaling, were critical for the cells to survive. Previous studies have shown that Netrin signaling helps the nervous system form in embryos and inhibits a program of controlled cell death in some cancers. Perampalam et al. discovered that Netrins were present in the environment immediately surrounding dormant HGSOC cells. Human HGSOC patients with higher levels of Netrin gene expression had poorer prognoses than patients with lower levels of Netrin gene expression. Further experiments demonstrated that Netrins help dormant HGSOC cells to spread around the body. These findings suggest that Netrin signalling may provide useful targets for future drug therapies against dormant cells in some ovarian cancers. This could include repurposing drugs already in development or creating new inhibitors of this pathway.

Fine tuning of CpG spatial distribution with DNA origami for improved cancer vaccination.

Multivalent presentation of ligands often enhances receptor activation and downstream signalling. DNA origami offers a precise nanoscale spacing of ligands, a potentially useful feature for therapeutic nanoparticles. Here we use a square-block DNA origami platform to explore the importance of the spacing of CpG oligonucleotides. CpG engages Toll-like receptors and therefore acts to activate dendritic cells. Through in vitro cell culture studies and in vivo tumour treatment models, we demonstrate that square blocks induce Th1 immune polarization when CpG is spaced at 3.5 nm. We observe that this DNA origami vaccine enhances DC activation, antigen cross-presentation, CD8 T-cell activation, Th1-polarized CD4 activation and natural-killer-cell activation. The vaccine also effectively synergizes with anti-PD-L1 for improved cancer immunotherapy in melanoma and lymphoma models and induces long-term T-cell memory. Our results suggest that DNA origami may serve as a platform for controlling adjuvant spacing and co-delivering antigens in vaccines.

Nesca, a novel neuronal adapter protein, links the molecular motor kinesin with the pre-synaptic membrane protein, syntaxin-1, in hippocampal neurons.

Vesicular transport in neurons plays a vital role in neuronal function and survival. Nesca is a novel protein that we previously identified and herein describe its pattern of expression, subcellular localization and protein-protein interactions both in vitro and in vivo. Specifically, a large proportion of Nesca is in tight association with both actin and microtubule cytoskeletal proteins. Nesca binds to F-actin, microtubules, ßIII and acetylated α-tubulin, but not neurofilaments or the actin-binding protein drebrin, in in vitro-binding assays. Nesca co-immunoprecipitates with kinesin heavy chain (KIF5B) and kinesin light-chain motors as well as with the synaptic membrane precursor protein, syntaxin-1, and is a constituent of the post-synaptic density. Moreover, in vitro-binding assays indicate that Nesca directly binds KIF5B, kinesin light-chain and syntaxin-1. In contrast, Nesca does not co-immunoprecipitate with the kinesin motors KIF1B, KIF3A nor does it bind syntaxin-4 or the synaptosome-associated protein 25 kDa (SNAP-25) in vitro. Nesca expression in neurons is highly punctuate, co-stains with syntaxin-1, and is found in fractions containing markers of early endosomes and Golgi suggesting that it is involved in vesicular transport. Collectively, these data suggest that Nesca functions as an adapter involved in neuronal vesicular transport including vesicles containing soluble N-ethylmaleimide sensitive factor attachment protein receptors that are essential to exocytosis.

Single-molecule mechanical fingerprinting with DNA nanoswitch calipers.

Decoding the identity of biomolecules from trace samples is a longstanding goal in the field of biotechnology. Advances in DNA analysis have substantially affected clinical practice and basic research, but corresponding developments for proteins face challenges due to their relative complexity and our inability to amplify them. Despite progress in methods such as mass spectrometry and mass cytometry, single-molecule protein identification remains a highly challenging objective. Towards this end, we combine DNA nanotechnology with single-molecule force spectroscopy to create a mechanically reconfigurable DNA nanoswitch caliper capable of measuring multiple coordinates on single biomolecules with atomic resolution. Using optical tweezers, we demonstrate absolute distance measurements with ångström-level precision for both DNA and peptides, and using multiplexed magnetic tweezers, we demonstrate quantification of relative abundance in mixed samples. Measuring distances between DNA-labelled residues, we perform single-molecule fingerprinting of synthetic and natural peptides, and show discrimination, within a heterogeneous population, between different posttranslational modifications. DNA nanoswitch calipers are a powerful and accessible tool for characterizing distances within nanoscale complexes that will enable new applications in fields such as single-molecule proteomics.

Nerve growth factor activation of the TrkA receptor induces cell death, by macropinocytosis, in medulloblastoma Daoy cells.

Ectopic expression of the TrkA receptor tyrosine kinase in tumors of the nervous system can mediate nerve growth factor (NGF)-dependent cell death by apoptosis and /or autophagy. Herein, we demonstrate that TrkA can also induce cell death in medulloblastoma Daoy cells by a caspase-independent mechanism that involves the hyperstimulation of macropinocytosis. Specifically, NGF-stimulates the uptake of AlexaFluor546-dextran into lysosome-associated membrane protein-1 positive vacuoles which fuse with microtubule associated protein light chain 3 (LC3) positive autophagosomes, to form large intracellular vacuoles (> 1 mum), which then fuse with lysotracker positive lysosomes. While LC3 cleavage and the appearance of LC3 positive vacuoles suggest the induction of autophagy, siRNA reduced expression of four proteins essential to autophagy (beclin-1, Atg5, LC3 and Atg9) neither blocks NGF-induced vacuole formation nor cell death. TrkA activated cell death does not require p38, JNK or Erk1/2 kinases but does require activation of class III PI-3 kinase and is blocked by the casein kinase 1 (CK1) inhibitor, D4476. This inhibitor does not interfere with TrkA activation but does block NGF-dependent AlexaFluor546-dextran uptake and CK1 dependent phosphorylation of beta-catenin. Collectively, these data demonstrate that TrkA stimulates cell death by a novel mechanism involving CK1-dependent hyperstimulation of macropinocytosis.

The fibroblast growth factor receptor substrate 3 adapter is a developmentally regulated microtubule-associated protein expressed in migrating and differentiated neurons.

Fibroblast growth factor (FGF) mediated signaling is essential to many aspects of neural development. Activated FGF receptors signal primarily through the FGF receptor substrate (Frs) adapters, which include Frs2/Frs2alpha and Frs3/Frs2beta. While some studies suggest that Frs3 can compensate for the loss of Frs2 in transfected cells, the lack of an effective Frs3 specific antibody has prevented efforts to determine the role(s) of the endogenous protein. To this end, we have generated a Frs3 specific antibody and have characterized the pattern of Frs3 expression in the developing nervous system, its subcellular localization as well as its biochemical properties. We demonstrate that Frs3 is expressed at low levels in the ventricular zone of developing cortex, between E12 and E15, and it co-localizes with nestin and acetylated alpha-tubulin in radial processes in the ventricular/subventricular zones as well as with betaIII tubulin in differentiated cortical neurons. Subcellular fractionation studies demonstrate that endogenous Frs3 is both soluble and plasma membrane associated while Frs3 expressed in 293T cells associates exclusively with lipid rafts. Lastly, we demonstrate that neuronal Frs3 binds microtubules comparable to the microtubule-associated protein, MAP2, while Frs2 does not. Collectively, these data suggest that neuronal Frs3 functions as a novel microtubule binding protein and they provide the first biochemical evidence that neuronal Frs3 is functionally distinct from Frs2/Frs2alpha.

C-Aryl glycoside inhibitors of SGLT2: Exploration of sugar modifications including C-5 spirocyclization.

Modifications to the sugar portion of C-aryl glycoside sodium glucose transporter 2 (SGLT2) inhibitors were explored, including systematic deletion and modification of each of the glycoside hydroxyl groups. Based on results showing activity to be quite tolerant of structural change at the C-5 position, a series of novel C-5 spiro analogues was prepared. Some of these analogues exhibit low nanomolar potency versus SGLT2 and promote urinary glucose excretion (UGE) in rats. However, due to sub-optimal pharmacokinetic parameters (in particular half-life), predicted human doses did not meet criteria for further advancement.

Nesca, a novel adapter, translocates to the nuclear envelope and regulates neurotrophin-induced neurite outgrowth.

We provide the first characterization of a novel signaling adapter, Nesca, in neurotrophic signal transduction. Nesca contains a RUN domain, a WW domain, a leucine zipper, a carboxyl-terminal SH3 domain, and several proline-rich regions. Nesca is highly expressed in the brain, is serine phosphorylated, and mobilizes from the cytoplasm to the nuclear membrane in response to neurotrophin, but not epidermal growth factor, stimulation in a MEK-dependent process. Overexpression studies in PC12 cells indicate that Nesca facilitates neurotrophin-dependent neurite outgrowth at nonsaturating doses of nerve growth factor (NGF). Similarly, short interfering RNA studies significantly reduce NGF-dependent neuritogenesis in PC12 cells. Mutational analyses demonstrate that the RUN domain is an important structural determinant for the nuclear translocation of Nesca and that the nuclear redistribution of Nesca is essential to its neurite outgrowth-promoting properties. Collectively, these works provide the first functional characterization of Nesca in the context of neurotrophin signaling and suggest that Nesca serves a novel, nuclear-dependent role in neurotrophin-dependent neurite outgrowth.

Trk receptor binding and neurotrophin/fibroblast growth factor (FGF)-dependent activation of the FGF receptor substrate (FRS)-3.

We have investigated the signaling properties of the fibroblast growth factor (FGF) receptor substrate 3 (FRS3), also known as SNT-2 or FRS2beta, in neurotrophin-dependent differentiation in comparison with the related adapter FRS2 (SNT1 or FRS2alpha). We demonstrate that FRS3 binds all neurotrophin Trk receptor tyrosine kinases and becomes tyrosine phosphorylated in response to NGF, BDNF, NT-3 and FGF stimulation in transfected cells and/or primary cortical neurons. Second, the signaling molecules Grb2 and Shp2 bind FRS3 at consensus sites that are highly conserved among FRS family members and that Shp2, in turn, becomes tyrosine phosphorylated. While FRS3 over-expression in PC12 cells neither increases NGF-induced neuritogenesis nor activation of Map kinase/AKT, comparable to previous reports on FRS2, over-expression of a chimeric adapter containing the PH/PTB domains of the insulin receptor substrate (IRS) 2, in place of the PTB domain of FRS3 (IRS2-FRS3) supports insulin-dependent Map kinase activation and neurite outgrowth in PC12 cells. Collectively, these data demonstrate that FRS3 supports ligand-induced Map kinase activation and that the chimeric IRS2-FRS3 adapter is stimulating sufficient levels of activated MapK to support neurite outgrowth in PC12 cells.

N-terminal specific conjugation of extracellular matrix proteins to 2-pyridinecarboxaldehyde functionalized polyacrylamide hydrogels.

Polyacrylamide hydrogels have been used extensively to study cell responses to the mechanical and biochemical properties of extracellular matrix substrates. A key step in fabricating these substrates is the conjugation of cell adhesion proteins to the polyacrylamide surfaces, which typically involves nonspecifically anchoring these proteins via side-chain functional groups. This can result in a loss of presentation control and altered bioactivity. Here, we describe a new functionalization strategy in which we anchor full-length extracellular matrix proteins to polyacrylamide substrates using 2-pyridinecarboxaldehyde, which can be co-polymerized into polyacrylamide gels and used to immobilize proteins by their N-termini. This one-step reaction proceeds under mild aqueous conditions and does not require additional reagents. We demonstrate that these substrates can readily conjugate to various extracellular matrix proteins, as well as promote cell adhesion and spreading. Notably, this chemistry supports the assembly and cellular remodeling of large collagen fibers, which is not observed using conventional side-chain amine-conjugation chemistry.

Unravelling the Mechanism of TrkA-Induced Cell Death by Macropinocytosis in Medulloblastoma Daoy Cells.

Macropinocytosis is a normal cellular process by which cells internalize extracellular fluids and nutrients from their environment and is one strategy that Ras-transformed pancreatic cancer cells use to increase uptake of amino acids to meet the needs of rapid growth. Paradoxically, in non-Ras transformed medulloblastoma brain tumors, we have shown that expression and activation of the receptor tyrosine kinase TrkA overactivates macropinocytosis, resulting in the catastrophic disintegration of the cell membrane and in tumor cell death. The molecular basis of this uncontrolled form of macropinocytosis has not been previously understood. Here, we demonstrate that the overactivation of macropinocytosis is caused by the simultaneous activation of two TrkA-mediated pathways: (i) inhibition of RhoB via phosphorylation at Ser(185) by casein kinase 1, which relieves actin stress fibers, and (ii) FRS2-scaffolded Src and H-Ras activation of RhoA, which stimulate actin reorganization and the formation of lamellipodia. Since catastrophic macropinocytosis results in brain tumor cell death, improved understanding of the mechanisms involved will facilitate future efforts to reprogram tumors, even those resistant to apoptosis, to die.

Neutralizing intraspinal nerve growth factor with a trkA-IgG fusion protein blocks the development of autonomic dysreflexia in a clip-compression model of spinal cord injury.

Increased intraspinal nerve growth factor (NGF) after spinal cord injury (SCI) is detrimental to the autonomic nervous system. Autonomic dysreflexia is a debilitating condition characterized by episodic hypertension, intense headache, and sweating. Experimentally, it is associated with aberrant primary afferent sprouting in the dorsal horn that is nerve growth factor (NGF)-dependent. Therapeutic strategies that neutralize NGF may ameliorate initial apoptotic cellular responses to the injury and aberrant afferent plasticity that occurs weeks after the injury. Subsequently, the development of autonomic disorders may be suppressed. We constructed a protein including the extracellular portion of trkA fused to the Fc portion of human IgG and expressed it using a baculovirus system. Binding of our trkA-IgG fusion protein was specific for NGF with a K(d) = 4.26 x 10(-11) M and blocked NGF-dependent neuritogenesis in PC-12 cells. We hypothesized that binding of NGF in the injured cord by our trkA-IgG fusion protein would diminish autonomic dysreflexia. Severe, high thoracic SCI was induced with clip compression and the rats were treated with intrathecal infusions (4 microg/day) of trkA-IgG or control IgG. At 14 days post-SCI, the magnitude of autonomic dysreflexia was assessed. Colon distension increased mean arterial pressure (MAP) in control rats by 46 +/- 2 from 96 +/- 5 mmHg. In contrast, MAP of rats treated with trkA-IgG increased by only 30 +/- 2 mmHg. Likewise, the MAP response to cutaneous stimulation was also reduced in rats treated with trkA-IgG (20 +/- 1 vs. 29 +/- 2). In contrast, trkA-IgG treatment had no effect on heart rate responses during colon distension or cutaneous stimulation. These results indicate that treatment with trkA-IgG to block NGF suppresses the development of autonomic dysreflexia after a clinically relevant spinal cord injury.

A retinoblastoma allele that is mutated at its common E2F interaction site inhibits cell proliferation in gene-targeted mice.

The retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1(ΔG)) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1(ΔG/ΔG) mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1(ΔG/ΔG) MEFs display a magnitude of E2F target gene derepression similar to that of Rb1(-/-) cells, even though Rb1(ΔG/ΔG) cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1(ΔG/ΔG) MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G1 to arrest proliferation. Some Rb1(ΔG/ΔG) mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non-E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified.

Loss of the mammalian DREAM complex deregulates chondrocyte proliferation.

Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes.

Ras guanine nucleotide releasing factor 1 (RasGrf1) enhancement of Trk receptor-mediated neurite outgrowth requires activation of both H-Ras and Rac.

We previously demonstrated that the guanine nucleotide exchange factor, RasGrf1, binds nerve growth factor (NGF)-activated TrkA and facilitates neurotrophin-induced neurite outgrowth in PC12 cells. RasGrf1 can activate both Ras and Rac, via intrinsic Cdc25 and DH domains, respectively, suggesting that the activation of both could contribute to this process. Previous studies have assayed constitutive neurite outgrowth following RasGrf1 over-expression in PC12 cells, in either the absence or presence of ectopic H-Ras, and have suggested an essential role for either Ras or Rac depending on the presence of H-Ras over-expression. In contrast, in this study, we have addressed the mechanism of how RasGrf1 facilitates neurite outgrowth in response to the neurotrophins, NGF and BDNF. Using Ras/Rac activation assays and site-directed RasGrf1 mutants, we find that both Ras and Rac are essential to neurotrophin-induced neurite outgrowth. Moreover, we find that H-Ras over-expression rescues the loss of neurite outgrowth observed with a Rac minus mutant and that RasGrf1 differentially stimulates NGF-dependent activation of Rac or Ras, depending on cell type. Collectively, these studies clarify the mechanism of how RasGrf1 expression facilitates neurotrophin-induced neurite outgrowth. Moreover, they suggest that H-Ras over-expression should be used with caution to measure phenotypic responses.

Posttranslational modifications of the retinoblastoma tumor suppressor protein as determinants of function.

The retinoblastoma tumor suppressor protein (pRB) plays an integral role in G1-S checkpoint control and consequently is a frequent target for inactivation in cancer. The RB protein can function as an adaptor, nucleating components such as E2Fs and chromatin regulating enzymes into the same complex. For this reason, pRB's regulation by posttranslational modifications is thought to be critical. pRB is phosphorylated by a number of different kinases such as cyclin dependent kinases (Cdks), p38 MAP kinase, Chk1/2, Abl, and Aurora b. Although phosphorylation of pRB by Cdks has been extensively studied, activities regulated through phosphorylation by other kinases are just starting to be understood. As well as being phosphorylated, pRB is acetylated, methylated, ubiquitylated, and SUMOylated. Acetylation, methylation, and SUMOylation play roles in pRB mediated gene silencing. Ubiquitinylation of pRB promotes its degradation and may be used to regulate apoptosis. Recent proteomic data have revealed that pRB is posttranslationally modified to a much greater extent than previously thought. This new information suggests that many unknown pathways affect pRB regulation. This review focuses on posttranslational modifications of pRB and how they influence its function. The final part of the review summarizes new phosphorylation sites from accumulated proteomic data and discusses the possibilities that might arise from this data.