The estrogen receptor ␣ (ER), a member of the steroid receptor superfamily, contains an N-terminal hormone-independent transcriptional activation function (AF-1) and a C-terminal hormone-dependent transcriptional activation function (AF-2). Here, we used in-gel kinase assays to determine that pp90 rsk1 activated by either epidermal growth factor (EGF) or phorbol myristate acetate specifically phosphorylates Ser-167 within AF-1. In vitro kinase assays demonstrated that pp90 rsk1 phosphorylates the N terminus of the wild-type ER but not of a mutant ER in which Ser-167 was replaced by Ala. In vivo, EGF stimulated phosphorylation of Ser-167 as well as Ser-118. Ectopic expression of active pp90 rsk1 increased the level of phosphorylation of Ser-167 compared to that of either a mutant pp90 rsk1 , which is catalytically inactive in the N-terminal kinase domain, or to that of vector control. The ER formed a stable complex with the mutant pp90 rsk1 in vivo. Transfection of the mutant pp90 rsk1 depressed ER-dependent transcription of both a wild-type ER and a mutant ER that had a defective AF-2 domain (ER TAF-1). Furthermore, replacing either Ser-118 or Ser-167 with Ala in ER TAF-1 showed similar decreases in transcription levels. A double mutant in which both Ser-118 and Ser-167 were replaced with Ala demonstrated a further decrease in transcription compared to either of the single mutations. Taken together, our results strongly suggest that pp90 rsk1 phosphorylates Ser-167 of the human ER in vivo and that Ser-167 aids in regulating the transcriptional activity of AF-1 in the ER.

To better understand how skeletal muscle myosin molecules move actin filaments, we determine the motion-generating biochemistry of a single myosin molecule and study how it scales with the motion-generating biochemistry of an ensemble of myosin molecules. First, by measuring the effects of various ligands (ATP, ADP, and P(i)) on event lifetimes, tau(on), in a laser trap, we determine the biochemical kinetics underlying the stepwise movement of an actin filament generated by a single myosin molecule. Next, by measuring the effects of these same ligands on actin velocities, V, in an in vitro motility assay, we determine the biochemistry underlying the continuous movement of an actin filament generated by an ensemble of myosin molecules. The observed effects of P(i) on single molecule mechanochemistry indicate that motion generation by a single myosin molecule is closely associated with actin-induced P(i) dissociation. We obtain additional evidence for this relationship by measuring changes in single molecule mechanochemistry caused by a smooth muscle HMM mutation that results in a reduced P(i)-release rate. In contrast, we observe that motion generation by an ensemble of myosin molecules is limited by ATP-induced actin dissociation (i.e., V varies as 1/tau(on)) at low [ATP], but deviates from this relationship at high [ATP]. The single-molecule data uniquely provide a direct measure of the fundamental mechanochemistry of the actomyosin ATPase reaction under a minimal load and serve as a clear basis for a model of ensemble motility in which actin-attached myosin molecules impose a load.

ATP hydrolysis drives actin filament dynamics. The effect known as treadmilling arises from the preferential addition of ATP-actin monomers to the plus or barbed end of actin and the preferential dissociation of ADP-actin from the minus or pointed end. Hydrolysis of ATP occurs after the monomer is incorporated into the filament. In addition, actin-binding proteins often have significantly different affinities for the ADPbound versus ATP-bound forms of actin. Both of these lines of evidence strongly suggest that there must be significant structural changes in actin induced by ATP hydrolysis. Indirect solution techniques such as proteolytic digestion rates and fluorescence studies (reviewed in Ref. 1) are in agreement with conformational differences between the ADP and ATP states, but direct structural evidence has been lacking until recently.Based on crystal structures of a tetramethylrhodaminelabeled monomeric actin (TMR-actin) 2 with ADP (2) or AMP-PNP (3) at the active site, Dominguez and co-workers proposed the provocative idea that ATP hydrolysis initiates a series of changes originating at the active site, that ultimately cause a loop-to-helix transition in the DNase binding loop in subdomain 2 of actin ("D-loop," residues in subdomain 2 of actin which comprise part of the DNase I binding site). They suggested that this was the long sought after change in structure between ADP and ATP actin. The cleft between subdomains 2 and 4 of actin remained closed in both nucleotide states. Despite this observation, an opposing point of view (4) held that the more important conformational change is an opening of the cleft upon ATP hydrolysis, a change that would be compatible with that observed for other nucleotide hydrolyzing proteins. Docking of actin crystal structures into negatively stained images of F-actin was also consistent with the idea that ADPactin had a more open cleft than the triphosphate state (5). If the latter view is correct, one would have to suppose that the modification of Cys 374 by TMR stabilized the closed conformation and inactivated the nucleotide sensing mechanism by virtue of the binding of rhodamine between subdomains 1 and 3, a potential hinge region (6) of the molecule. It has been suggested (4) that the short helix observed in the crystal structure of the ADP state of TMR-actin could be formed as a result of contacts with neighboring molecules in the crystal. We provide further evidence to support this idea, and suggest a pathway through which nucleotide-dependent changes may propagate through fortuitous crystal packing interactions from one monomer to the D-loop region of another in the TMR-actin crystals.Here we crystallized and expressed a cytoplasmic actin that was rendered incapable of polymerization by virtue of two surface mutations in subdomain 4 (A204E/P243K). This strategy negates concerns raised about the TMR modification of actin.De novo crystallization of AP-actin with either ATP or ADP at the active site reveals obligatory nucleotide-dependent confor-* This work was suppor...

Phosphorylation of Ser 118 of human estrogen receptor ␣ (ER) enhances ER-mediated transcription and is in

Serine 118 is definitively identified as a major site of phosphorylation in the human estrogen receptor expressed in COS-1 cells treated with estradiol or phorbol ester. At least 30% of the estrogen receptor appears to be phosphorylated on serine 118 after treatment with estradiol or phorbol ester. Human estrogen receptor was expressed in COS-1 cells and labeled in vivo with [32P]orthophosphate in the presence of estradiol or phorbol ester. Immunopurified receptor was digested with cyanogen bromide. The most heavily labeled peptide (7 kilodaltons) was identified as amino acids 110-174 by microsequencing. Manual Edman degradation released a major portion of the 32P-label in the peptide at serine 118. A mutant with serine 118 replaced by alanine (S118A) had 80% less 32P-label in the 7 kilodalton peptide. Estrogen receptor labeled in vivo with [32P]-orthophosphate in the presence of estradiol or phorbol ester migrates electrophoretically as a doublet. The major difference between the bands is phosphorylation of serine 118 in the upshifted band. The mutant S118A does not show an upshifted band. Labeling of the estrogen receptor with [35S]methionine indicates that > or = 30% of the receptor is upshifted and suggests that > or = 30% of the receptor is phosphorylated on serine 118.

Actin stimulates myosin's activity by inducing structural alterations that correlate with the transition from a weakly to a strongly bound state, during which time inorganic phosphate (P i ) is released from myosin's active site. The surface loop at the 50/20-kDa junction of myosin (loop 2) is part of the actin interface. Here we demonstrate that elimination of two highly conserved lysines at the C-terminal end of loop 2 specifically blocks the ability of heavy meromyosin to undergo a weak to strong binding transition with actin in the presence of ATP. Removal of these lysines has no effect on strong binding in the absence of nucleotide, on the rate of ADP binding or release, or on the basal ATPase activity. We further show that the 16 amino acids of loop 2 preceding the lysine-rich region are not essential for actin activation, although they do modulate myosin's affinity for actin in the presence of ATP. We conclude that interaction of the conserved lysines with acidic residues in subdomain 1 of actin either triggers a structural change or stabilizes a conformation that is necessary for actinactivated release of P i and completion of the ATPase cycle.The generation of force and movement by the myosin-actin interaction results from an ATP-driven cycle that alternates between dissociated states, weakly bound actomyosin states, and strongly bound actomyosin complexes. This cyclic interaction between actin and myosin is thought to involve the steps illustrated in Scheme 1, where A is actin and M is myosin.

The fungal toxin cytochalasin D (CD) interferes with the normal dynamics of the actin cytoskeleton by binding to the barbed end of actin filaments. Despite its widespread use as a tool for studying actin-mediated processes, the exact location and nature of its binding to actin has not been previously determined. Here we describe two crystal structures of an expressed monomeric actin in complex with CD, one obtained by soaking preformed actin crystals with CD, and the other by cocrystallization. The binding site for CD, in the hydrophobic cleft between actin subdomains 1 and 3, is the same in the two structures. Polar and hydrophobic contacts play an equally important role in CD binding, and six hydrogen bonds stabilize the actin-CD complex. Many unrelated actin-binding proteins and marine toxins target this cleft, and the hydrophobic pocket at the front end of the cleft (viewing actin with subdomain 2 in the upper right corner). CD differs in that it binds to the back half of the cleft. The ability of CD to induce actin dimer formation and actin-catalyzed ATP hydrolysis may be related to its unique binding site, and the necessity to fit its bulky macrocycle into this cleft. Contacts with residues lining this cleft appear to be crucial to capping and/or severing. The cocrystallized actin-CD structure also revealed changes in actin conformation. A rotation of ~6° of the smaller actin domain (subdomains 1 and 2) with respect to the larger domain (subdomains 3 and 4) results in small changes in crystal packing that allow the D-loop to adopt an extended loop structure, instead of being disordered as it is in most crystal structures of actin. We speculate that these changes represent a potential conformation that the actin monomer can adopt on the pathway to polymerization or in the filament.

We have succeeded in expressing actin in the baculovirus/Sf9 cell system in high yield. The wild-type (WT) actin is functionally indistinguishable from tissue-purified actin in its ability to activate ATPase activity and to support movement in an in vitro motility assay. Having achieved this feat, we used a mutational strategy to express a monomeric actin that is incapable of polymerization. Native actin requires actin binding proteins or chemical modification to maintain it in a monomeric state. The mutant actin sediments in the analytical ultracentrifuge as a homogeneous monomeric species of 3.2 S in 100 mM KCl and 2 mM MgCl(2), conditions that cause WT actin to polymerize. The two point mutations that render actin nonpolymerizable are in subdomain 4 (A204E/P243K; "AP-actin"), distant from the myosin binding site. AP-actin binds to skeletal myosin subfragment 1 (S1) and forms a homogeneous complex as demonstrated by analytical ultracentrifugation. The ATPase activity of a cross-linked AP-actin.S1 complex is higher than that of S1 alone, although less than that supported by filamentous actin (F-actin). AP-Actin is an excellent candidate for structural studies of complexes of actin with motor proteins and other actin-binding proteins.