Abrogation of retinoblastoma protein function by c-Abl through tyrosine kinase-dependent and -independent mechanisms.

The decision to enter the cell division cycle is governed by the interplay between growth activators and growth inhibitors. The retinoblastoma protein (RB) is an example of a growth inhibitor whose main function appears to be the binding and inactivation of key cell cycle activators. One target of RB is a proto-oncoprotein, the c-Abl tyrosine kinase. RB binds to the ATP-binding lobe in the kinase domain and inhibits the nuclear pool of c-Abl in quiescent and G1 cells. Phosphorylation of RB at G1/S releases c-Abl, leading to the activation of this nuclear tyrosine kinase. In this report, we describe the construction of a mutant Abl, replacing the ATP-binding lobe of c-Abl with that of c-Src. The mutant protein AS2 is active as a tyrosine kinase and can phosphorylate Abl substrates, such as the C-terminal repeated domain of RNA polymerase II. AS2, however, does not bind to RB, and its activity is not inhibited by RB. As a result, the nuclear pool of AS2 is no longer cell cycle regulated. Excess AS2, but not its kinase-defective counterpart, can overcome RB-induced growth arrest in Saos-2 cells. Interestingly, wild-type c-Abl, in both its kinase-active and -inactive forms, can also overcome RB. Furthermore, overexpression of a kinase-defective c-Abl in rodent fibroblasts accelerates the transition from quiescence to S phase and cooperates with c-Myc to induce transformation. These effects, however, do not occur with the kinase-defective form of AS2. Thus, the growth-stimulating function of the kinase-defective c-Abl is dependent on the binding and the abrogation of RB function. That RB function can be abolished by the overproduction of one of its binding proteins is consistent with the hypothesis that RB induces cell cycle arrest by acting as a "molecular matchmaker" to assemble protein complexes. Exclusive engagement of RB by one of its many targets is incompatible with the biological function of this growth suppressor protein.     

How the other half lives, the amino-terminal domain of the retinoblastoma tumor suppressor protein

The retinoblastoma tumor suppressor gene (RB1) is currently the only known gene whose mutation is necessary and sufficient for the development of a human cancer. Mutation or deregulation of RB1 is observed so frequently in other tumor types that compromising RB1 function may be a prerequisite for malignant transformation. Identifying the molecular mechanisms that provide the basis for RB1-mediated tumor suppression has become an important goal in the quest to understand and treat cancer. The lion's share of research on these mechanisms has focused on the carboxy-terminal half of the RB1 encoded protein (pRB). This focus is with good reason since this part of the protein, now called the "large pocket," is required for most of its known activities identified in vitro and in vivo. Large pocket mediated mechanisms alone, however, cannot account for all observed properties of pRB. The thesis presented here is that the relatively uncharacterized amino-terminal half of the protein makes important contributions to pRB-mediated tumor suppression. The goals of this review are to summarize evidence indicating that an amino-terminal structural domain is important for pRB function and to suggest a general hypothesis as to how this domain can be integrated with current models of pRB function.     

Purification and characterization of a functionally homogeneous 60-kDa species of the retinoblastoma gene product.

The retinoblastoma susceptibility gene (RB) encodes a 928-amino acid protein (pRB) that is hypothesized to function in a pathway that restricts cell proliferation. The immortalizing proteins from three distinct DNA tumor viruses (SV40 large T antigen, adenovirus E1a, and human papilloma virus Type 16 E7) have been shown to interact with RB protein through two noncontiguous regions comprised of amino acids 393-572 (domain A) and 646-772 (domain B). We constructed a truncated form of RB (RB p60) that retains these two domains but eliminates the N-terminal 386 amino acids of RB. RB p60 was expressed in Escherichia coli in inclusion bodies. After solubilization, it was refolded in the presence of magnesium chloride, and the active protein was isolated with an E7 peptide affinity column. The protein that elutes from this column is functionally homogenous in its ability to bind immobilized E7 protein. Thermal denaturation studies provide additional evidence for the conformational homogeneity of the isolated protein. This purification scheme allows the isolation of significant amounts of RB p60 protein that is suitable for structural and functional studies.     

Actomyosin cross-linking by caldesmon in non-muscle cells

The role of myosin-binding in cytoskeletal arrangement of non-muscle low molecular weight caldesmon (l-caldesmon) was studied. The N-terminal myosin-binding domain of caldesmon N152 colocalized with myosin in transiently transfected chicken fibroblasts. When added exogenously to the Triton-insoluble cytoskeleton, N152 enhanced l-caldesmon displacement by exogenous C-terminal actin-binding fragment (H1). Thus, a significant fraction of l-caldesmon cross-links actin and myosin. In contrast, in epithelioid HeLa cells most of l-caldesmon was only actin-bound as H1 alone was enough for its displacement. Phosphorylation by mitogen-activated protein kinase reduced the capability of H1 to displace endogenous l-caldesmon, suggesting it may represent a regulatory mechanism for actin-caldesmon interaction in vivo.     

The 14-3-3 protein affects the conformation of the regulatory domain of human tyrosine hydroxylase

Tyrosine hydroxylase (TH) catalyzes the first step in the biosynthesis of catecholamines. Regulation of TH enzyme activity is controlled through the posttranslational modification of its regulatory domain. The regulatory domain of TH can be phosphorylated at four serines (8, 19, 31, and 40) by a variety of protein kinases. Phosphorylation of Ser19 does not by itself increase TH activity but induces its binding to the 14-3-3 protein. That leads to the enhancement of TH activity with a still not fully understood mechanism. The main goal of this work was to investigate whether the 14-3-3 protein binding affects the conformation of the regulatory domain of human TH isoform 1 (TH1R). Site-directed mutagenesis was used to generate five single-tryptophan mutants of TH1R with the Trp residue located at five different positions within the domain (positions 14, 34, 73, 103, and 131). Time-resolved tryptophan fluorescence measurements revealed that phosphorylation of Ser19 and Ser40 does not itself induce any significant structural changes in regions surrounding inserted tryptophans. On the other hand, the interaction between the 14-3-3 protein and phosphorylated TH1R decreases the solvent exposure of tryptophan residues at positions 14 and 34 and induces distinct structural change in the vicinity of Trp73. The 14-3-3 protein binding also reduces the sensitivity of phosphorylated TH1R to proteolysis by protecting its N-terminal part (first 33 residues). Circular dichroism measurements showed that TH1R is an unstructured protein with a low content of secondary structure and that neither phosphorylation nor the 14-3-3 protein binding changes its secondary structure.     

Superhelical architecture of the myosin filament-linking protein myomesin with unusual elastic properties

Active muscles generate substantial mechanical forces by the contraction/relaxation cycle, and, to maintain an ordered state, they require molecular structures of extraordinary stability. These forces are sensed and buffered by unusually long and elastic filament proteins with highly repetitive domain arrays. Members of the myomesin protein family function as molecular bridges that connect major filament systems in the central M-band of muscle sarcomeres, which is a central locus of passive stress sensing. To unravel the mechanism of molecular elasticity in such filament-connecting proteins, we have determined the overall architecture of the complete C-terminal immunoglobulin domain array of myomesin by X-ray crystallography, electron microscopy, solution X-ray scattering, and atomic force microscopy. Our data reveal a dimeric tail-to-tail filament structure of about 360 Å in length, which is folded into an irregular superhelical coil arrangement of almost identical α-helix/domain modules. The myomesin filament can be stretched to about 2.5-fold its original length by reversible unfolding of these linkers, a mechanism that to our knowledge has not been observed previously. Our data explain how myomesin could act as a highly elastic ribbon to maintain the overall structural organization of the sarcomeric M-band. In general terms, our data demonstrate how repetitive domain modules such as those found in myomesin could generate highly elastic protein structures in highly organized cell systems such as muscle sarcomeres.     

Molecular dynamic simulations of the N-terminal receiver domain of NtrC reveal intrinsic conformational flexibility in the inactive state

The N-terminal receiver domain of NtrC is the molecular switch in the two-component signal transduction. It is the first protein where structures of both the active (phosphyroylated) and inactive (unphosphyroylated) states are determined experimentally. Phosphorylation of the NtrC at the active site induces large structural change. NMR experiments suggested that the wild type unphosphorylated NtrC adopts both the active and the inactive conformations and the phosphorylation stabilizes the active conformations. We applied free (unconstrained) molecular dynamic (MD) simulation to examine the intrinsic flexibilities and stabilities of the NtrC receiver domain in both the active and inactive conformations. Molecular dynamic simulations showed that the inactive state of NtrC receiver domain is more flexible than the active state. There were large movements in helix 4 and loop beta3-alpha3 which coincide with major structural differences between the inactive and active states. We observed large root-mean-square deviations from the initial starting structure and the large root-mean-square fluctuations during MD simulation for the inactive state. We then investigated the activation pathway with Targeted MD simulation. We show that the intrinsic flexibility in the loop beta3-alpha3 plays an important role in triggering the conformational change. Phosphorylation at the active site may serve to stabilize the conformational change. These results together suggest that the unphosphorylated NtrC receiver domain could be involved in a conformational equilibrium between two different states.     

The regions of the retinoblastoma protein needed for binding to adenovirus E1A or SV40 large T antigen are common sites for mutations

The protein product of the retinoblastoma (RB) gene is thought to function in a pathway that restricts cell proliferation. Recently, transforming proteins from three different classes of DNA tumor viruses have been shown to form complexes with the RB protein. Genetic studies suggest that these interactions with the RB protein are important steps in transformation by these viruses. In order to understand better the function of the RB-viral oncoprotein complexes, we have mapped the regions of the RB protein that are necessary for these associations. Two non-contiguous regions of RB were found to be essential for complex formation with adenovirus E1A or SV40 large T antigen. These two regions are found between amino acids 393 and 572 and 646 and 772. Interestingly, these binding sites on RB overlap with the positions of naturally occurring, inactivating mutations of the RB gene. These results strongly suggest that these viral oncoproteins are targeting a protein domain that is an important site in the normal function of the RB protein.     

Temperature-Sensitive RB Mutations Linked to Incomplete Penetrance of Familial Retinoblastoma in 12 Families

The tumor-suppressor activity of the retinoblastoma protein (RB) is encoded within a protein-binding ("pocket") domain that is targeted for mutations in all cases of familial retinoblastoma and in many common adult cancers. Although familial retinoblastoma is a paradigm for a highly penetrant, recessive model of tumorigenesis, the molecular basis for the phenotype of incomplete penetrance of familial retinoblastoma is undefined. We studied the RB pocket-binding properties of three independent, mutant RB alleles that are present in the germline of 12 kindreds with the phenotype of incomplete penetrance of familial retinoblastoma. Each arises from alterations of single codons within the RB pocket domain (designated "delta 480," "661W," or "712R"). Under the same conditions, we studied the properties of wild-type (WT) RB, an RB point mutant isolated from a lung carcinoma sample (706F) and an adjacent, in vitro-generated point mutant (707W). The delta 480, 661W, and 712R mutants lack pocket protein-binding activity in vitro but retain the WT ability to undergo cyclin-mediated phosphorylation in vivo. Each of the low-penetrant RB mutants exhibits marked enhancement of pocket protein binding when the cells are grown at reduced temperature. In contrast, in this temperature range, no change in binding activity is seen with WT RB, the 706F mutant, or the 707W mutant. We have demonstrated that many families with incomplete penetrance of familial retinoblastoma carry unstable, mutant RB alleles with temperature-sensitive pocket protein-binding activity. The variable frequency for tumor development in these families may result from reversible fluctuations in a threshold level of RB pocket-binding activity.     

The F-BAR domain of SRGP-1 facilitates cell-cell adhesion during C. elegans morphogenesis

Robust cell-cell adhesion is critical for tissue integrity and morphogenesis, yet little is known about the molecular mechanisms controlling cell-cell junction architecture and strength. We discovered that SRGP-1 is a novel component of cell-cell junctions in Caenorhabditis elegans, localizing via its F-BAR (Bin1, Amphiphysin, and RVS167) domain and a flanking 200-amino acid sequence. SRGP-1 activity promotes an increase in membrane dynamics at nascent cell-cell contacts and the rapid formation of new junctions; in addition, srgp-1 loss of function is lethal in embryos with compromised cadherin-catenin complexes. Conversely, excess SRGP-1 activity leads to outward bending and projections of junctions. The C-terminal half of SRGP-1 interacts with the N-terminal F-BAR domain and negatively regulates its activity. Significantly, in vivo structure-function analysis establishes a role for the F-BAR domain in promoting rapid and robust cell adhesion during embryonic closure events, independent of the Rho guanosine triphosphatase-activating protein domain. These studies establish a new role for this conserved protein family in modulating cell-cell adhesion.     

Kinetic basis for the stimulatory effect of phosphorylation on the methylesterase activity of CheB

Response regulators are activated to elicit a specific cellular response to an extracellular stimulus via phosphotransfer from a cognate sensor histidine kinase to a specific aspartate residue. Phosphorylation at the conserved aspartate residue modulates the activity of the response regulator. Methylesterase CheB is a two-domain response regulator composed of a regulatory domain and an effector domain with enzymatic activity. CheB functions within the bacterial chemotaxis pathway to control the level of chemoreceptor methylation. In its unphosphorylated state, the regulatory domain inhibits methylesterase activity of the effector domain. Phosphorylation of the regulatory domain leads to an enhancement of methylesterase activity through a relief of inhibition and a stimulatory effect on catalysis. CheB is a useful model protein for understanding the effects of phosphorylation of the regulatory domain on interdomain interactions and stimulation of enzymatic activity of the effector domain. Kinetic analyses of CheB activation indicate that the basis for the nearly 100-fold methylesterase activation upon phosphorylation is due to a change in the catalytic rate constant for the methylesterase reaction. It is also shown that the P2 domain of histidine kinase CheA inhibits the methylesterase activity of CheB and that this inhibition is decreased upon phosphorylation of CheB. Finally, studies of methylesterase catalysis by the free catalytic domain in the presence and absence of the regulatory domain have enabled detection of an association between the two domains in the absence of the linker.     

Tumors with nonfunctional retinoblastoma protein are killed by reduced γ-tubulin levels

In various tumors inactivation of growth control is achieved by interfering with the RB1 signaling pathway. Here, we describe that RB1 and γ-tubulin proteins moderate each other's expression by binding to their respective gene promoters. Simultaneous reduction of RB1 and γ-tubulin protein levels results in an E2F1-dependent up-regulation of apoptotic genes such as caspase 3. We report that in various tumors types, there is an inverse correlation between the expression levels of γ-tubulin and RB1 and that in tumor cell lines with a nonfunctioning RB1, reduction of γ-tubulin protein levels leads to induction of apoptosis. Thus, the RB1/γ-tubulin signal network can be considered as a new target for cancer treatment.     

Molecular Dynamic Simulations of the N-Terminal Receiver Domain of NtrC Reveal Intrinsic Conformational Flexibility in the Inactive State

Abstract

The N-terminal receiver domain of NtrC is the molecular switch in the two-component signal transduction. It is the first protein where structures of both the active (phosphyroylated) and inactive (unphosphyroylated) states are determined experimentally. Phosphorylation of the NtrC at the active site induces large structural change. NMR experiments suggested that the wild type unphosphorylated NtrC adopts both the active and the inactive conformations and the phosphorylation stabilizes the active conformations. We applied free (unconstrained) molecular dynamic (MD) simulation to examine the intrinsic flexibilities and stabilities of the NtrC receiver domain in both the active and inactive conformations. Molecular dynamic simulations showed that the inactive state of NtrC receiver domain is more flexible than the active state. There were large movements in helix 4 and loop β3-α3 which coincide with major structural differences between the inactive and active states. We observed large root-mean-square deviations from the initial starting structure and the large root-mean-square fluctuations during MD simulation for the inactive state. We then investigated the activation pathway with Targeted MD simulation. We show that the intrinsic flexibility in the loop β3-α3 plays an important role in triggering the conformational change. Phosphorylation at the active site may serve to stabilize the conformational change. These results together suggest that the unphosphorylated NtrC receiver domain could be involved in a conformational equilibrium between two different states.     

Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation

Introduction of an exogenous retinoblastoma (RB) gene in RB-deficient retinoblastoma or osteosarcoma cells has been shown to suppress their neoplastic phenotype. In experiments designed to explore the potential mechanism of RB tumor suppression, we report here that the phosphorylation state of RB protein is modulated during normal cellular events. In resting cells, RB protein is present in its least phosphorylated form; in rapidly proliferating cells, RB protein is highly phosphorylated. Maximal phosphorylation is associated with S phase of the cell cycle. Induction of differentiation in several human leukemia cell lines by treatment with phorbol ester or retinoic acid leads to dephosphorylation of RB. Time course studies indicate that RB dephosphorylation precedes the total arrest of cell growth during differentiation. These observations strongly suggest that the function of RB protein is modulated by a phosphorylation/dephosphorylation mechanism during cell proliferation and differentiation.     

Molecular basis of the head‐to‐tail assembly of giant muscle proteins obscurin‐like 1 and titin

Large filament proteins in muscle sarcomeres comprise many immunoglobulin‐like domains that provide a molecular platform for self‐assembly and interactions with heterologous protein partners. We have unravelled the molecular basis for the head‐to‐tail interaction of the carboxyl terminus of titin and the amino‐terminus of obscurin‐like‐1 by X‐ray crystallography. The binary complex is formed by a parallel intermolecular β‐sheet that presents a novel immunoglobulin‐like domain‐mediated assembly mechanism in muscle filament proteins. Complementary binding data show that the assembly is entropy‐driven rather than dominated data by specific polar interactions. The assembly observed leads to a V‐shaped zipper‐like arrangement of the two filament proteins.     

Phosphorylated Grb14 is an endogenous inhibitor of retinal protein tyrosine phosphatase 1B, and light-dependent activation of Src phosphorylates Grb14

Growth factor receptor-bound protein 14 (Grb14) is an adapter protein implicated in receptor tyrosine kinase signaling. Grb14(-/-) studies highlight both the positive and negative roles of Grb14 in receptor tyrosine kinase signaling in a tissue-specific manner. In this study, we made a novel finding that Grb14 inhibits the activity of PTP1B, the major negative regulator of insulin receptor (IR) signaling, in a phosphorylation-regulated manner. Phosphorylation of Tyr-347 in the BPS domain of Grb14 is critical for interaction with PTP1B, resulting in the competitive inhibition of PTP1B activity. We also found that rhodopsin-regulated Src kinase activation in retina leads to the phosphorylation of Grb14. Further, ablation of Grb14 resulted in significantly elevated retinal PTP1B activity in vivo. PTP1B is known to be regulated by oxidation, glutathionylation, phosphorylation, and SUMOlyation, and our study for the first time demonstrates the inhibition of PTP1B activity in vivo by protein molecule Grb14 in a tissue-specific manner.