Mutant Chimeras Research Articles

An Atomic Level Investigation of Sodium Ions Regulating Agonist and Antagonist Binding in the Active Site of a Novel Target 5HT2BR Against Drug-Resistant Epilepsy.

The study investigates the movement of sodium ions inside the ligand-binding pocket of the class-A GPCR serotonin receptor (5HT2BR), a primary target for modern drugs. The available PDBs are mutant chimeras, so a 3D structure is modeled and validated by structural similarity (84.05%), Ramachandran favorable residues (93.01%), and clash score. Using MD simulations (500 ns), the ion active site is tracked in the presence and absence of ions and ligands. The ions enter the active site along helices III, VI, and VII, and the primary residue (ASP3.32) interacts with ions via H-bond (stronger- ~2.4 Å). The radial distribution function around ASP3.32 rises promptly at intermediate distances (2 Å < r < 4 Å), suggesting a higher probability of finding sodium ions at these distances. The ions stabilize the receptor at a better RMSD and promote stronger interactions (3-H-bonds, 1-π-bond~3.35 Å) with the agonist, and not the antagonist (no H-bond). Simulating unrestrained ligands further confirms this pattern, suggesting that ions might promote agonist binding but not be a prerequisite for antagonist action. The study highlights the mechanistic evaluation of sodium ions mobility in 5HT2BR modulation and ligand binding, showing potential in drug development.

Differences in the 3' intergenic region and the V2 protein of two sequence variants of tomato curly stunt virus play an important role in disease pathology in Nicotiana benthamiana.

Tomato production in South Africa is threatened by the emergence of tomato curly stunt virus (ToCSV), a monopartite Begomovirus transmitted by the whitefly vector Bemisia tabaci (Genn.). We investigated the role of sequence differences present in the 3' intergenic region (IR) and the V2 coding region on the differing infectivity of ToCSV sequence variant isolates V30 and V22 in the model host Nicotiana benthamiana. Using virus mutant chimeras, we determined that the development of the upward leaf roll symptom phenotype is mediated by sequence differences present in the 3' IR containing the TATA-associated composite element. Sequence differences present in the V2 coding region are responsible for modulating disease severity and symptom recovery in V22-infected plants. Serine substitution of V22 V2 Val27 resulted in a significant increase in disease severity with reduced recovery, the first study to demonstrate the importance of this V2 residue in disease development. Two putative ORFs, C5 and C6, were identified using in silico analysis and detection of an RNA transcript spanning their coding region suggests that these ORFs may be transcribed during infection. Additional virus-derived RNA transcripts spanning multiple ORFs and crossing the boundaries of recognised polycistronic transcripts, as well as the origin of replication within the IR, were detected in ToCSV-infected plants providing evidence of bidirectional readthrough transcription. From our results, we conclude that the diverse responses of the model host to ToCSV infection is influenced by select sequence differences and our findings provide several avenues for further investigation into the mechanisms behind these responses to infection.

Construction of functional chimeras of syntaxin-1A and its yeast orthologue, and their application to the yeast cell-based assay for botulinum neurotoxin serotype C

BackgroundBotulinum neurotoxins (BoNTs) prevent synaptic transmission because they hydrolyze synaptic N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). BoNT serotype C (BoNT/C) targets syntaxin-1A and SNAP-25, and is expected to be applied to cosmetic and therapeutic uses. SNAREs are evolutionally conserved proteins and in yeast a syntaxin-1A orthologue Sso1 is involved in exocytosis. The substrate specificity of BoNT/C is strict and it cannot cleave Sso1. MethodsDomain swapping and mutational screenings were performed to generate functional chimeras SNAREs of syntaxin-1A and Sso1. Such chimeras are expressed in yeast cells and assessed whether they are susceptible to BoNT/C digestion. ResultsThe Sso1 and syntaxin-1A chimera (Sso1/STX1A), in which the SNARE domain in Sso1 was replaced with that of syntaxin-1A, was not functional in yeast. The functional incompatibility of Sso1/STX1A was attributable to its accumulation in the ER. We found several mutations that could release Sso1/STX1A from the ER to make the chimera functional in yeast. Yeast cells harboring the mutant chimeras grew similarly to wild-type cells. However, unlike wild-type, yeast harboring the mutant chimeras exhibited a severe growth defect upon expression of BoNT/C. Results of further domain swapping analyses suggest that Sso1 is not digested by BoNT/C because it lacks a binding region to BoNT/C (α-exosite-binding region). ConclusionsWe obtained functional Sso1/STX1A chimeras, which can be applied to a yeast cell-based BoNT/C assay. BoNT/C can recognize these chimeras in a similar manner to syntaxin-1A. General significanceThe yeast cell-based BoNT/C assay would be useful to characterize and engineer BoNT/C.

Cryopreservation by encapsulation-dehydration affects the vegetative growth of chrysanthemum but does not disturb its chimeric structure

Little is known about the stability of plant chimeras after exposure to liquid nitrogen. The aim of this study was to evaluate the effect of cryopreservation on the cytogenetic and genetic stability, as well as vegetative and generative growth of chrysanthemums that are a solid mutant (‘Richmond’) or periclinal chimeras (‘Lady Orange’ and ‘Lady Salmon’). The following encapsulation-dehydration protocol of shoot tip cryopreservation was applied: 0.09 M sucrose concentration and 10 µM abscisic acid during a two-week preculture, followed by a 4-day osmotic dehydration and 3-hour desiccation. Both, the cryopreservation-recovered and control plants were characterized by the same relative DNA content and chromosome number (ploidy level). By applying RAPD and ISSR markers, 18 and 1 and 0 polymorphic loci within cryopreservation-derived ‘Lady Orange’ and ‘Lady Salmon’ and ‘Richmond’ were detected, respectively. The recovered after cryopreservation and control plants had the same colour, diameter and fresh weight of inflorescences, as well as length of ray florets. Cryopreservation also did not affect the flowering time in chrysanthemum. The biochemical assay did not reveal any alternations in the level of anthocyanins and carotenoids in flowers. It was noted, however, that some of the leaves of the cryopreservation-recovered plants were shorter and/or narrower. They had a reduced chlorophyll content, and their internodes were shorter when compared to the untreated control. The inflorescences of ‘Lady Salmon’ opened slower, but faded faster. In conclusion, these results illustrate the practicability of a cryopreservation method that completely protects the chimera chrysanthemum identity.

Phosphoinositide 3-OH Kinase Regulates Integrin-Dependent Processes in Neutrophils by Signaling through Its Effector ARAP3

ARAP3, a GTPase activating protein for Rho and Arf family GTPases, is one of many phosphoinositide 3-OH kinase (PI3K) effectors. In this study, we investigate the regulatory input of PI3K upstream of ARAP3 by analyzing neutrophils from an ARAP3 pleckstrin homology (PH) domain point mutation knock-in mouse (R302, 303A), in which ARAP3 is uncoupled from activation by PI3K. ARAP3 PH domain point mutant neutrophils are characterized by disturbed responses linked to stimulation by either integrin ligands or immobilized immune complexes. These cells exhibit increased β2 integrin inside-out signaling (binding affinity and avidity), and our work suggests the disturbed responses to immobilized immune complexes are secondary to this. In vitro, neutrophil chemotaxis is affected in the mutant. In vivo, ARAP3 PH domain point mutant bone marrow chimeras exhibit reduced neutrophil recruitment to the peritoneum on induction of sterile peritonitis and also reduced inflammation in a model for rheumatoid arthritis. The current work suggests a dramatic regulatory input of PI3K into the regulation of β2 integrin activity, and processes dependent on this, by signaling through its effector ARAP3.

Two distinct knockout approaches highlight a critical role for p53 in rat development

Gene targeting in embryonic stem cells (ESCs) has become the principal technology for generating knockout models. Although numerous studies have predicted that the disruption of p53 leads to increased developmental anomalies and malignancies, most p53 knockout mice develop normally. Therefore, the role of p53 in animal development was examined using rat knockout models. Conventionally generated homozygous KO males developed normally, whereas females rarely survived due to neural tube defects. Mutant chimeras generated via blastocyst injection with p53-null ESCs exhibited high rates of embryonic lethality in both sexes. This phenotype could be observed in one month by the use of zinc-finger nucleases. The p53-null ESCs were resistant to apoptosis and differentiation, and exhibited severe chromosome instabilities in the chimera-contributed cells, suggesting an essential role for p53 in maintaining ESC quality and genomic integrity. These results demonstrate that p53 functions as a guardian of embryogenesis in the rats.

Acute depletion of plasma membrane phospholipids—dissecting the roles of PtdIns(4)P and PtdIns(4,5)P2

Acute depletion of plasma membrane phospholipids—dissecting the roles of PtdIns(4)P and PtdIns(4,5)P2

Differential Requirement for Utrophin in the Induced Pluripotent Stem Cell Correction of Muscle versus Fat in Muscular Dystrophy Mice

Duchenne muscular dystrophy (DMD) is an incurable degenerative muscle disorder. We injected WT mouse induced pluripotent stem cells (iPSCs) into mdx and mdx∶utrophin mutant blastocysts, which are predisposed to develop DMD with an increasing degree of severity (mdx <<< mdx∶utrophin). In mdx chimeras, iPSC-dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Dystrobrevin was observed in dystrophin-positive and, at a lesser extent, utrophin-positive areas. In the mdx∶utrophin mutant chimeras, although iPSC-dystrophin was also supplied to the muscle sarcolemma, mice still displayed poor skeletal muscle histopathology, and negligible levels of dystrobrevin in dystrophin- and utrophin-negative areas. Not only dystrophin-expressing tissues are affected by iPSCs. Mdx and mdx∶utrophin mice have reduced fat/body weight ratio, but iPSC injection normalized this parameter in both mdx and mdx∶utrophin chimeras, despite the fact that utrophin was compromised in the mdx∶utrophin chimeric fat. The results suggest that the presence of utrophin is required for the iPSC-corrections in skeletal muscle. Furthermore, the results highlight a potential (utrophin-independent) non-cell autonomous role for iPSC-dystrophin in the corrections of non-muscle tissue like fat, which is intimately related to the muscle.

Inhibition of pituitary tumors in Rb mutant chimeras through E2f4 loss reveals a key suppressive role for the pRB/E2F pathway in urothelium and ganglionic carcinogenesis

The retinoblastoma protein pRB suppresses tumorigenesis largely through regulation of the E2F transcription factors. E2F4, the most abundant E2F protein, is thought to act in cooperation with pRB to restrain cell proliferation. In this study, we analyze how loss of E2f4 affects the tumorigenicity of pRB-deficient tissues. Since Rb-/-;E2f4-/- germline mice die in utero, we generated Rb-/-;E2f4-/- chimeric animals to allow examination of adult tumor phenotypes. We found that loss of E2f4 had a differential effect on known Rb-associated neuroendocrine tumors. It did not affect thyroid and adrenal glands tumors but partially suppressed lung neuroendocrine hyperplasia. The most striking effect was in the pituitary where E2F4-loss delayed the development, and reduced the incidence, of Rb mutant tumors. This tumor suppression increased the longevity of the Rb-/-;E2f4-/- chimeric animals allowing us to identify novel tumor types. We observed ganglionic neuroendocrine neoplasms, lesions not previously associated with mutation of either Rb or E2f4. Moreover, a subset of the Rb-/-;E2f4-/- chimeras developed either low or high-grade carcinomas in the urothelium transitional epithelium supporting a key role for Rb in bladder cancer.

Analysis of the Thermodynamics of Binding of an SH3 Domain to Proline-rich Peptides using a Chimeric Fusion Protein

A complete understanding of the thermodynamic determinants of binding between SH3 domains and proline-rich peptides is crucial to the development of rational strategies for designing ligands for these important domains. Recently we engineered a single-chain chimeric protein by fusing the α-spectrin Src homology region 3 (SH3) domain to the decapeptide APSYSPPPPP (p41). This chimera mimics the structural and energetic features of the interaction between SH3 domains and proline-rich peptides. Here we show that analysing the unfolding thermodynamics of single-point mutants of this chimeric fusion protein constitutes a very useful approach to deciphering the thermodynamics of SH3–ligand interactions. To this end, we investigated the contribution of each proline residue of the ligand sequence to the SH3–peptide interaction by producing six single Pro-Ala mutants of the chimeric protein and analysing their unfolding thermodynamics by differential scanning calorimetry (DSC). Structural analyses of the mutant chimeras by circular dichroism, fluorescence and NMR together with NMR-relaxation measurements indicate conformational flexibility at the binding interface, which is strongly affected by the different Pro-Ala mutations. An analysis of the DSC thermograms on the basis of a three-state unfolding model has allowed us to distinguish and separate the thermodynamic magnitudes of the interaction at the binding interface. The model assumes equilibrium between the “unbound” and “bound” states at the SH3–peptide binding interface. The resulting thermodynamic magnitudes classify the different proline residues according to their importance in the interaction as P2∼P7∼P10 > P9∼P6 > P8, which agrees well with Lim's model for the interaction between SH3 domains and proline-rich peptides. In addition, the thermodynamic signature of the interaction is the same as that usually found for this type of binding, with a strong enthalpy–entropy compensation for all the mutants. This compensation appears to derive from an increase in conformational flexibility concomitant to the weakening of the interactions at the binding interface. We conclude that our approach, based on DSC and site-directed mutagenesis analysis of chimeric fusion proteins, may serve as a suitable tool to analyse the energetics of weak biomolecular interactions such as those involving SH3 domains.

The Actin-depolymerizing Factor Homology and Charged/Helical Domains of Drebrin and mAbp1 Direct Membrane Binding and Localization via Distinct Interactions with Actin

Cytoskeletal dynamics are important for efficient function of the secretory pathway. ADP-ribosylation factor, ARF1, triggers vesicle coat assembly and, in concert with Cdc42, regulates actin polymerization and molecular motor-based motility. Drebrin and mammalian Abp1 (mAbp1) are actin-binding proteins found previously to bind to Golgi membranes in an ARF1-dependent manner in vitro. Despite sharing homology through two shared actin binding domains, drebrin and mAbp1 have different subcellular localization and bind to distinct actin structures on the Golgi apparatus. We find that the actin-depolymerizing factor homology (ADFH) and charged/helical actin binding domains of drebrin and mAbp1 are sufficient for regulated binding to Golgi membranes and subcellular localization. We have used mutant proteins and chimeras between mAbp1 and drebrin to identify motifs that direct targeting. We find that a linker region between the ADFH and charged/helical domains confers Golgi binding properties to mAbp1. mAbp1 binds to a specific actin pool through its ADFH/linker domain that is not bound by drebrin. Drebrin localization to the cell surface was found to involve motifs within the charged/helical domain. Our results indicate that targeting of these proteins is directed through multiple distinct interactions with the actin cytoskeleton. The mechanisms for selective recruitment of mAbp1 and drebrin to Golgi membranes indicate how actin-based structures are able to select specific actin-binding proteins and, thus, carry out multiple different functions within cells.

Design of a fluorescence-activated cell sorting-based Mammalian protein-protein interaction trap.

The mammalian protein-protein interaction trap (MAPPIT) is a two-hybrid assay based on insights in type I cytokine signal transduction. Bait and prey polypeptides are tethered to mutant cytokine receptor chimeras which are impaired in signaling. On bait-prey interaction and after ligand stimulation, the JAK-STAT signaling cascade is initiated leading to transcription of a reporter or marker gene under the control of the STAT3-responsive rPAP1 promoter. In addition to a physiologically relevant context for mammalian protein-protein interactions this method provides separation of interactor and effector zones, and can be applied for both analytical and screening purposes. In the protocol described here, a cytokine receptor derived surface tag is used as a selectable marker. After an initial presort step using magnetic-activated cell sorting (MACS), "positive" cells are selected by fluorescence-activated cell sorting (FACS).

Wild-type nonneuronal cells extend survival of SOD1 mutant motor neurons in ALS mice.

The most common inherited [correct] form of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting adult motor neurons, is caused by dominant mutations in the ubiquitously expressed Cu-Zn superoxide dismutase (SOD1). In chimeric mice that are mixtures of normal and SOD1 mutant-expressing cells, toxicity to motor neurons is shown to require damage from mutant SOD1 acting within nonneuronal cells. Normal motor neurons in SOD1 mutant chimeras develop aspects of ALS pathology. Most important, nonneuronal cells that do not express mutant SOD1 delay degeneration and significantly extend survival of mutant-expressing motor neurons.

The domain order of mammalian capping enzyme can be inverted and baculovirus phosphatase can function in cap formation in vivo.

The bifunctional mammalian mRNA capping enzyme (Mce1) consists of an N-terminal triphosphatase domain Mce1(1-210) fused to a C-terminal guanylyltransferase domain Mce1(211-597). The physical domain order H(2)N-triphosphatase-guanylyltransferase-COOH mimics the temporal order of the capping reactions. To determine if the physical domain order is functionally important in vivo, we engineered an "inverted" mammalian capping enzyme InvMce1 [H(2)N-Mce1(211-597)-(1-210)-COOH]. We found that InvMce1 complemented the growth of Saccharomyces cerevisiae cet1delta and ceg1delta strains in which the endogenous yeast triphosphatase and guanylyltransferase genes were deleted. By testing truncated versions of InvMce1, we determined that Mce1(1-178) comprises a minimal functional triphosphatase domain. Baculovirus phosphatase (BVP) is a monofunctional single-domain protein with RNA triphosphatase and RNA diphosphatase activities and an undefined role in viral RNA metabolism. Here we demonstrated that BVP can function as an RNA triphosphatase for cap formation in vivo when fused to the C-terminus of Mce1(211-597). By characterizing a series of InvMce1-BVP derivatives with amino acid substitutions in the phosphate-binding loop of BVP, we showed that the in vivo activity of the mutant chimeras in cap formation is contingent upon in vitro phosphohydrolase activity of the respective BVP proteins. BVP catalysis in vitro was not limited to 5'-phosphorylated RNA or nucleotide substrates, but also embraced tripolyphosphatase and pyrophosphatase activities. BVP-specific activities with nucleotide and inorganic substrates were as follows: ATP (14 min(-1)), ADP (31 min(-1)), PPP(i) (3.7 min(-1)), and PP(i) (1 min(-1)). BVP did not hydrolyze AMP. We surmise that BVP has adapted the cysteinyl phosphatase fold to the hydrolysis of phosphoanhydrides.

Deficiency of phospholipase C-gamma1 impairs renal development and hematopoiesis.

Phospholipase C-gamma1 (PLC-gamma1) is involved in a variety of intracellular signaling via many growth factor receptors and T-cell receptor. To explore the role of PLC-gamma1 in vivo, we generated the PLC-gamma1-deficient (plc-gamma1(-/-)) mice, which died of growth retardation at embryonic day 8.5-9.5 in utero. Therefore, we examined plc-gamma1(-/-) chimeric mice generated with plc-gamma1(-/-) embryonic stem (ES) cells for further study. Pathologically, plc-gamma1(-/-) chimeras showed multicystic kidney due to severe renal dysplasia and renal tube dilation. Flow cytometric analysis and glucose phosphate isomerase assay revealed very few hematopoietic cells derived from the plc-gamma1(-/-) ES cells in the mutant chimeras. However, differentiation of plc-gamma1(-/-) ES cells into erythrocytes and monocytes/macrophages in vitro was observed to a lesser extent compared with control wild-type ES cells. These data suggest that PLC-gamma1 plays an essential role in the renal development and hematopoiesis in vivo.

Thermostabilization of a chimeric enzyme by residue substitutions: four amino acid residues in loop regions are responsible for the thermostability of Thermus thermophilus isopropylmalate dehydrogenase

A chimeric 3-isopropylmalate dehydrogenase, named 2T2M6T, made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, was found to be considerably more labile than the T. thermophilus wild-type isopropylmalate dehydrogenase. In order to identify the molecular basis of the thermal stability of the T. thermophilus isopropylmalate dehydrogenase, 11 amino acid residues in the mesophilic portion of the chimera were substituted by the corresponding residues of the T. thermophilus enzyme, and the effects of the side chain substitutions were analyzed by comparing the reaction rate of irreversible heat denaturation and catalytic parameters of the mutant chimeras with those of the original chimera, 2T2M6T. Four single-site mutants were successfully stabilized without any loss of the catalytic function. All these four sites are located in loop regions of the enzyme. Our results strongly suggest the importance of these loop structures to the extreme stability of the T. thermophilus isopropylmalate dehydrogenase.

Communication between myocytes and fibroblasts in cardiac remodeling in angiotensin chimeric mice.

To characterize the mode of action of angiotensin II (Ang II) in cardiac remodeling, we generated chimeric mice that are made of both homozygous Ang II receptor type 1A gene (Agtr1a) null mutant cells and Agtr1a intact cells expressing the lacZ gene (ROSA26). Both Agtr1a null and intact myocytes and interstitial cells independently form areas that are randomly distributed throughout the heart. The distribution of ROSA26 cardiomyocytes overlaps completely with that of Ang II binding, indicating that the majority of Ang II receptors reside on cardiomyocytes. When Ang II (1 ng/g body weight/min) was infused for 2 weeks, mice developed mild to moderate hypertension. The proliferating cardiac fibroblasts identified by bromodeoxyuridine staining were present predominantly in the areas surrounded by Agtr1a intact cardiomyocytes. When control chimeric mice made of wild-type cells and ROSA26 cells (i.e., both carrying intact Agtr1a) were infused with Ang II, fibroblast proliferation was found equally in these cardiomyocyte types. When compared with Agtr1a null mutant chimeras, the control chimeras had more extensive cardiac fibrosis, most prominently in perivascular regions. Therefore, in response to Ang II, cardiac fibroblasts proliferate through both the local and systemic action of Ang II. Importantly, the former is determined by the Ang II receptor of neighboring cardiomyocytes, indicating that a communication between myocytes and fibroblasts plays an important role during Ang II-dependent cardiac remodeling.

Mammalian Grb2 Regulates Multiple Steps in Embryonic Development and Malignant Transformation

Proteins with SH2 and SH3 domains link tyrosine kinases to intracellular pathways. To investigate the biological functions of a mammalian SH2/SH3 adaptor, we have introduced a null mutation into the mouse gene for Grb2. Analysis of mutant embryonic stem cells, embryos, and chimeras reveals that Grb2 is required during embyrogenesis for the differentiation of endodermal cells and formation of the epiblast. Grb2 acts physiologically as an adaptor, since replacing the C terminus of the Ras activator Sos1 with the Grb2 SH2 domain yields a fusion protein that largely rescues the defects caused by the Grb2 mutation. Furthermore, Grb2 is rate limiting for mammary carcinomas induced by polyomavirus middle T antigen. These data provide genetic evidence for a mammalian Grb2–Ras signaling pathway, mediated by SH2/SH3 domain interactions, that has multiple functions in embryogenesis and cancer.

Dystrophic Muscle in Mice Chimeric for Expression of α5 Integrin

alpha5-deficient mice die early in embryogenesis (). To study the functions of alpha5 integrin later in mouse embryogenesis and during adult life we generated alpha5 -/-;+/+ chimeric mice. These animals contain alpha5-negative and positive cells randomly distributed. Analysis of the chimerism by glucose- 6-phosphate isomerase (GPI) assay revealed that alpha5 -/- cells contributed to all the tissues analyzed. High contributions were observed in the skeletal muscle. The perinatal survival of the mutant chimeras was lower than for the controls, however the subsequent life span of the survivors was only slightly reduced compared with controls (). Histological analysis of alpha5 -/-;+/+ mice from late embryogenesis to adult life revealed an alteration in the skeletal muscle structure resembling a typical muscle dystrophy. Giant fibers, increased numbers of nuclei per fiber with altered position and size, vacuoli and signs of muscle degeneration-regeneration were observed in head, thorax and limb muscles. Electron microscopy showed an increase in the number of mitochondria in some muscle fibers of the mutant mice. Increased apoptosis and immunoreactivity for tenascin-C were observed in mutant muscle fibers. All the alterations were already visible at late stages of embryogenesis. The number of altered muscle fibers varied in different animals and muscles and was often increased in high percentage chimeric animals. Differentiation of alpha5 -/- ES cells or myoblasts showed that in vitro differentiation into myotubes was achieved normally. However proper adhesion and survival of myoblasts on fibronectin was impaired. Our data suggest that a novel form of muscle dystrophy in mice is alpha5-integrin-dependent.

Transcriptional Activation of Heat Shock Factor HSF1 Probed by Phosphopeptide Analysis of Factor 32P-labeled in Vivo

Mapping of tryptic phosphopeptides of heat shock factor 1 (HSF1) from non-stressed or moderately heat-stressed HeLa cells, labeled in vivo by [32P]orthophosphate, revealed four major phosphopeptides A to D. Heat stress drastically increased phosphopeptide signals. To identify target peptides and amino acids and to correlate phosphorylation and transactivation function, phosphopeptide maps were produced of LexA-human HSF1 chimeras and mutant derivatives thereof, and transactivation activities of original and mutant chimeras were compared. LexA-HSF1 chimeras were previously shown to be regulated identically to HSF1, except that they transactivate promoters with LexA-binding sites instead of hsp promoters. The patterns of phosphopeptides of LexA-HSF1 and endogenous HSF1 were similar. Analysis of single residue substitutions suggested that phosphopeptide C is peptide VKEEPPSPPQSPR (297-309) phosphorylated on Ser-307 but not Ser-303. Substitution of Ser-307 but not Ser-303 caused deregulation of factor activity. Mapping of several constitutively active chimeras associated unphosphorylated peptide C with the transcriptionally active HSF1 conformation, suggesting that dephosphorylation of this peptide (at Ser-307) may either be an integral step in the activation process or serve to maintain the active conformation of HSF1. Exploiting this correlation, indirect evidence was obtained that activation domains of HSF1 interact with the distantly located regulatory domain to maintain the factor in an inactive state.