Regulation of fragile X mental retardation 1 protein by C-terminus of Hsc70-interacting protein depends on its phosphorylation status

The fragile X mental retardation 1 (FMR1) protein binds mRNA and acts as a negative regulator of translation. Lack of FMR1 causes the most common neurological disorder, fragile X syndrome, while its overexpression is associated with metastasis of breast cancer. Its activity has been well-studied in nervous tissue, but recent evidence as well as its role in cancer indicates that it also acts in other tissues. We have investigated the expression of FMR1 in brain and other tissues of mouse and examined its regulation. We detected expression of FMR1 in liver and heart tissues of mice as well as in brain tissue, supporting other contentions that it acts in non-nervous tissue. Expression of FMR1 inversely correlated with expression of the C-terminus of Hsc70-interacting protein (CHIP) and, based on the known activity of CHIP in protein homeostasis, we suggest that CHIP regulates expression of FMR1. CHIP ubiquitinated FMR1 for proteasomal degradation in a molecular chaperone-independent manner. FMR1 expression was reduced following treatment with okadaic acid, a phosphatase inhibitor, but not in CHIP-depleted cells. Also, a non-phospho FMR1 mutant was much less efficiently ubiquitinated by CHIP and had a longer half-life compared to either wild-type FMR or a phospho-mimic mutant. Taken together, our results demonstrate that CHIP regulates the levels of FMR1 as an E3 ubiquitin ligase in phosphorylation-dependent manner, suggesting that CHIP regulates FMR1-mediated translational repression by regulating the levels of FMR1.     

Domain:domain interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for protein stability and structure

The major heat shock protein (Hsp) chaperones Hsp70 and Hsp90 both bind the co-chaperone Hop (Hsp70/Hsp90 organizing protein), which coordinates Hsp actions in folding protein substrates. Hop contains three tetratricopeptide repeat (TPR) domains that have binding sites for the conserved EEVD C termini of Hsp70 and Hsp90. Crystallographic studies have shown that EEVD interacts with positively charged amino acids in Hop TPR-binding pockets (called carboxylate clamps), and point mutations of these carboxylate clamp positions can disrupt Hsp binding. In this report, we use circular dichroism to assess the effects of point mutations and Hsp70/Hsp90 peptide binding on Hop conformation. Our results show that Hop global conformation is destabilized by single point mutations in carboxylate clamp positions at pH 5, while the structure of individual TPR domains is unaffected. Binding of peptides corresponding to the C termini of Hsp70 and Hsp90 alters the global conformation of wild-type Hop, whereas peptide binding does not alter conformation of individual TPR domains. These results provide biophysical evidence that Hop-binding pockets are directly involved with domain:domain interactions, both influencing Hop global conformation and Hsp binding, and contributing to proper coordination of Hsp70 and Hsp90 interactions with protein substrates.     

C-terminus of Hsc70-interacting protein regulates profilin1 and breast cancer cell migration

Profilin1 (Pfn1) is a key mediator of actin polymerization and regulates cell migration. Low expression of Pfn1 is implicated in tumorigenesis of various cancers, including breast cancer. The regulatory mechanism behind Pfn1 levels has not yet been elucidated. In the present study, we find that Pfn1 is poly-ubiquitinated in human cell lines, and a portion of poly-ubiquitinated Pfn1 is regulated in a proteasome-dependent manner. C-terminus of Hsc70-interacting protein (CHIP), a co-chaperone E3 ligase, interacts with and ubiquitinates Pfn1, targeting it for proteasome-dependent degradation. Depletion of CHIP stabilizes Pfn1, suggesting that CHIP functions as a major E3 ligase for Pfn1. Stable expression of wild-type CHIP in the breast cancer cell line MDA-MB231 yielded downregulation of Pfn1 and enhanced cell migration. Pfn1 overexpression in MDA-MB231 cells expressing wild-type CHIP suppressed the enhanced cell migration. Taken together, our results demonstrate that CHIP regulates Pfn1 levels as an E3 ligase, and possibly plays a role in cell migration and metastasis of breast cancer.     

Molecular cloning and characterization of an Hsp90/70 organizing protein gene from Frankliniella occidentalis (Insecta: Thysanoptera,Thripidae)

The heat shock 90/70 organizing protein (Hop), also known as Sti-1 (stress-induced protein-1), is a co-chaperone that usually mediates the interaction of Hsp90 and Hsp70 and has been extensively characterized in mammals and plants. However, its role in insects remains unknown. In the present study, we isolated and characterized a Hop homologue gene from Frankliniella occidentalis (Fohop). The Fohop contains a 1659 bp ORF encoding a protein of 552 amino acids with a caculated molecular mass of approximately 62.25 kDa, which displays a reasonable degree of identity with the known Hops and shares several canonical motifs, including three tetratricopeptide repeated motif domains (TPR1, TPR2A and TPR2B) and two aspartic acid–proline (DP) repeat motifs (DP1 and DP2). As in other hops, Fohop contains introns, but the number and the position are quite variable. The mRNA expression patterns indicated that Fohop was constitutively expressed throughout the developmental stages, but was obviously upregulated by heat stress both in larvae and adults. Our studies imply that Hop, as in other Hsps, may play an important role in heat shock response of F. occidentalis.     

U-box protein carboxyl terminus of Hsc70-interacting protein (CHIP) mediates poly-ubiquitylation preferentially on four-repeat Tau and is involved in neurodegeneration of tauopathy

Neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated and ubiquitylated tau, are exhibited at regions where neuronal loss occurs in neurodegenerative diseases; however, the mechanisms of NFT formation remain unknown. Molecular studies of frontotemporal dementia with parkinsonism-17 demonstrated that increasing the ratio of tau with exon 10 insertion induced fibrillar tau accumulation. Here, we show that carboxyl terminus of Hsc70-interacting protein (CHIP), a U-box protein, recognizes the microtubule-binding repeat region of tau and preferentially ubiquitylates four-repeat tau compared with three-repeat tau. Overexpression of CHIP induced the prompt degradation of tau, reduced the formation of detergent-insoluble tau and inhibited proteasome inhibitor-induced cell death. NFT bearing neurons in progressive supranuclear palsy, in which four-repeat tau is a component, showed the accumulation of CHIP. Thus, CHIP is a ubiquitin ligase for four-repeat tau and maintains neuronal survival by regulating the quality control of tau in neurons.     

Structural studies on the co-chaperone Hop and its complexes with Hsp90

The tetratricopeptide repeat domain (TPR)-containing co-chaperone Hsp-organising protein (Hop) plays a critical role in mediating interactions between Heat Shock Protein (Hsp)70 and Hsp90 as part of the cellular assembly machine. It also modulates the ATPase activity of both Hsp70 and Hsp90, thus facilitating client protein transfer between the two. Despite structural work on the individual domains of Hop, no structure for the full-length protein exists, nor is it clear exactly how Hop interacts with Hsp90, although it is known that its primary binding site is the C-terminal MEEVD motif. Here, we have undertaken a biophysical analysis of the structure and binding of Hop to Hsp90 using a variety of truncation mutants of both Hop and Hsp90, in addition to mutants of Hsp90 that are thought to modulate the conformation, in particular the N-terminal dimerisation of the chaperone. The results establish that whilst the primary binding site of Hop is the C-terminal MEEVD peptide of Hsp90, binding also occurs at additional sites in the C-terminal and middle domain. In contrast, we show that another TPR-containing co-chaperone, CyP40, binds solely to the C-terminus of Hsp90.Truncation mutants of Hop were generated and used to investigate the dimerisation interface of the protein. In good agreement with recently published data, we find that the TPR2a domain that contains the Hsp90-binding site is also the primary site for dimerisation. However, our results suggest that residues within the TPR2b may play a role. Together, these data along with shape reconstruction analysis from small-angle X-ray scattering measurements are used to generate a solution structure for full-length Hop, which we show has an overall butterfly-like quaternary structure.Studies on the nucleotide dependence of Hop binding to Hsp90 establish that Hop binds to the nucleotide-free, ‘open’ state of Hsp90. However, the Hsp90-Hop complex is weakened by the conformational changes that occur in Hsp90 upon ATP binding. Together, the data are used to propose a detailed model of how Hop may help present the client protein to Hsp90 by aligning the bound client on Hsp70 with the middle domain of Hsp90. It is likely that Hop binds to both monomers of Hsp90 in the form of a clamp, interacting with residues in the middle domain of Hsp90, thus preventing ATP hydrolysis, possibly by the prevention of association of N-terminal and middle domains in individual Hsp90 monomers.     

Identification of Hsc70 as an influenza virus matrix protein (M1) binding factor involved in the virus life cycle

Influenza virus matrix protein 1 (M1) has been shown to play a crucial role in the virus replication, assembly and budding. We identified heat shock cognate protein 70 (Hsc70) as a M1 binding protein by immunoprecipitation and MALDI-TOF MS. The C terminal domain of M1 interacts with Hsc70. We found that Hsc70 does not correlate with the transport of M1 to the nucleus, however, it does inhibit the nuclear export of M1 and NP, thus resulting in the inhibition of viral production. This is the first demonstration that Hsc70 is directly associated with M1 and therefore is required for viral production.     

S100 proteins regulate the interaction of Hsp90 with Cyclophilin 40 and FKBP52 through their tetratricopeptide repeats

S100 proteins are a subfamily of the EF-hand type calcium sensing proteins, the exact biological functions of which have not been clarified yet. In this work, we have identified Cyclophilin 40 (CyP40) and FKBP52 (called immunophilins) as novel targets of S100 proteins. These immunophilins contain a tetratricopeptide repeat (TPR) domain for Hsp90 binding. Using glutathione-S transferase pull-down assays and immunoprecipitation, we have demonstrated that S100A1 and S100A2 specifically interact with the TPR domains of FKBP52 and CyP40 in a Ca²⁺-dependent manner, and lead to inhibition of the CyP40-Hsp90 and FKBP52-Hsp90 interactions. These findings have suggested that the Ca²⁺/S100 proteins are TPR-targeting regulators of the immunophilins-Hsp90 complex formations.

Structured summary

MINT-7710442: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0915) with S100A6 (uniprotkb:P06703) by competition binding (MI:0405)MINT-7710192: Cyp40 (uniprotkb:P26882) binds (MI:0407) to S100A1 (uniprotkb:P35467) by pull down (MI:0096)MINT-7710412: Cyp40 (uniprotkb:P26882) physically interacts (MI:0915) with S100A2 (uniprotkb:P29034) by competition binding (MI:0405)MINT-7710374: FKBP52 (uniprotkb:Q02790) binds (MI:0407) to S100A2 (uniprotkb:P29034) by pull down (MI:0096)MINT-7710452: Cyp40 (uniprotkb:P26882) physically interacts (MI:0914) with S100A2 (uniprotkb:P29034) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710387: FKBP52 (uniprotkb:Q02790) binds (MI:0407) to S100A6 (uniprotkb:P06703) by pull down (MI:0096)MINT-7710279: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0915) with S100A1 (uniprotkb:P35467) by competition binding (MI:0405)MINT-7710224: FKBP52 (uniprotkb:Q02790) binds (MI:0407) to Hsp90 (uniprotkb:P07900) by pull down (MI:0096)MINT-7710464: Cyp40 (uniprotkb:P26882) physically interacts (MI:0914) with S100A6 (uniprotkb:P06703) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710249: Cyp40 (uniprotkb:P26882) binds (MI:0407) to Hsp90 (uniprotkb:P07900) by pull down (MI:0096)MINT-7710422: Cyp40 (uniprotkb:P26882) physically interacts (MI:0915) with S100A6 (uniprotkb:P06703) by competition binding (MI:0405)MINT-7710348: Cyp40 (uniprotkb:P26882) binds (MI:0407) to S100A2 (uniprotkb:P29034) by pull down (MI:0096)MINT-7710208: FKBP52 (uniprotkb:Q02790) binds (MI:0407) to S100A1 (uniprotkb:P35467) by pull down (MI:0096)MINT-7710265: Cyp40 (uniprotkb:P26882) physically interacts (MI:0915) with S100A1 (uniprotkb:P35467) by competition binding (MI:0405)MINT-7710361: Cyp40 (uniprotkb:P26882) binds (MI:0407) to S100A6 (uniprotkb:P06703) by pull down (MI:0096)MINT-7710476: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0914) with S100A2 (uniprotkb:P29034) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710316: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0914) with S100A1 (uniprotkb:P35467) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710432: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0915) with S100A2 (uniprotkb:P29034) by competition binding (MI:0405)MINT-7710488: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0914) with S100A6 (uniprotkb:P06703) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710329: S100A6 (uniprotkb:P14069) physically interacts (MI:0914) with FKBP52 (uniprotkb:P30416) and Cyp40 (uniprotkb:Q08752) by anti bait coimmunoprecipitation (MI:0006)MINT-7710295: Cyp40 (uniprotkb:P26882) physically interacts (MI:0914) with Hsp90 (uniprotkb:P07900) and S100A1 (uniprotkb:P35467) by anti tag coimmunoprecipitation (MI:0007)     

C. elegans STI-1, the Homolog of Sti1/Hop, Is Involved in Aging and Stress Response

Environmental and physiological stresses such as heat shock, oxidative stress, heavy metals, and pathogenic conditions induce cellular stress response. This response is often mediated by heat shock proteins that function as molecular chaperones. A stress-inducible cochaperone, Sti1/Hop (Hsp organizer protein), functions as an adaptor protein that simultaneously binds with Hsp70 and Hsp90 to transfer client proteins from Hsp70 to Hsp90. However, the biological role of STI-1 in vivo is poorly understood in metazoans. Here, we report the characterization of the Caenorhabditis elegans homolog of Sti1/Hop, which is approximately 56% identical with human STI-1. C. elegans STI-1 (CeSTI-1) is expressed in the pharynx, intestine, nervous system, and muscle from larvae to adults. Analysis of proteins immunoprecipitated with anti-STI-1 antibody by mass spectrometry revealed that CeSTI-1 can bind with both Hsp70 and Hsp90 homologs like its mammalian counterpart. sti-1 expression is elevated by heat stress, and an sti-1(jh125) null mutant shows decreased fertility under heat stress conditions. These mutants also show abnormally high lethality in extreme heat and may be functioning with DAF-16 in thermotolerance. In addition, sti-1(jh125) mutants have a shortened life span. Our results confirm that CeSTI-1 is a cochaperone protein that may maintain homeostatic functions during episodes of stress and can regulate longevity in nematodes.     

Interaction of mouse TTC30/DYF-1 with multiple intraflagellar transport complex B proteins and KIF17

Intraflagellar transport (IFT) is a microtubule based system that supports the assembly and maintenance of cilia. Genetic and biochemical studies have identified two distinct complexes containing multiple proteins that are part of the IFT machinery. In this study we prepared mouse pituitary cells that expressed an epitope-tagged IFT protein and immuno-purified the IFT B complex from these cells. Mass spectrometry analysis of the isolated complex led to identification of a number of well known components of the IFT B complex. In addition, peptides corresponding to mouse tetratricopeptide repeat proteins, TTC30A1, TTC30A2 and TTC30B were identified. The mouse Ttc30A1, Ttc30A2, Ttc30B genes are orthologs of Caenorhabditis elegans dyf-1, which is required for assembly of the distal segment of the cilia. We used co-immunoprecipitation studies to provide evidence that, TTC30A1, TTC30A2 or TTC30B can be incorporated into a complex with a known IFT B protein, IFT52. We also found that TTC30B can interact with mouse KIF17, a kinesin which participates in IFT. In vitro expression in a cell-free system followed by co-immunoprecipitation also provided evidence that TTC30B can directly interact with several different IFT B complex proteins. The findings support the view that mouse TTC30A1, TTC30A2 and TTC30B can contribute to the IFT B complex, likely through interactions with multiple IFT proteins and also suggest a possible link to the molecular motor, KIF17 to support transport of cargo during IFT.     

The Non-canonical Hop Protein from Caenorhabditis elegans Exerts Essential Functions and Forms Binary Complexes with Either Hsc70 or Hsp90

Heat shock protein (Hsp) 70/Hsp90-organizing proteins (Hop/Sti1) are thought to function as adaptor proteins to link the two chaperone machineries Hsp70 and Hsp90 during the processing of substrate proteins in eukaryotes. Hop (Hsp70/Hsp90-organizing protein) is composed of three tetratricopeptide repeat (TPR) domains, of which the first (TPR1) binds to Hsp70, the second (TPR2A) binds to Hsp90, and the third (TPR2B) is of unknown function. Contrary to most other eukaryotes, the homologue closest to the Caenorhabditis elegans Hop homologue R09E12.3 (CeHop) lacks the TPR1 domain and the short linker region connecting it to TPR2A, questioning the reported function as an Hsp90/Hsp70 adaptor in vitro and in vivo. We observed high constitutive expression levels of CeHop and detected significant phenotypes upon knockdown, linking the protein to functions in gonad development. Interestingly, we observed physical interactions with both chaperones Hsp70 and Hsp90, albeit only the interaction with Hsp90 is strong and inhibition of the Hsp90 ATPase activity can be observed upon binding of CeHop. However, the formation of ternary complexes with both chaperone machineries is impaired, as Hsp70 and Hsp90 compete for CeHop interaction sites, in particular as Hsp90 binds to both TPR domains simultaneously and requires both TPR domains for ATPase regulation. These results imply that, at least in C. elegans, essential functions of Hop exist which apparently do not depend on the simultaneous binding of Hsp90 and Hsp70 to Hop.     

The Assembly and Intermolecular Properties of the Hsp70-Tomm34-Hsp90 Molecular Chaperone Complex

Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding.     

Differential ubiquitination of Smad1 mediated by CHIP: implications in the regulation of the bone morphogenetic protein signaling pathway

Smad1, a downstream regulator of the bone morphogenetic protein (BMP) receptors, is tightly regulated by the ubiquitin-proteasomal degradation system. To dissect the mechanisms that underlie the regulation of Smad1, it is important to investigate the specific ubiquitination site(s) in Smad1. Here we report that the α-NH₂ group of the N terminus and the ε-NH₂ groups of internal lysine residues 116, 118 and 269 (K116, K118 and K269) of Smad1 are ubiquitin acceptor sites mediated by the carboxyl terminus of Hsc70-interacting protein (CHIP). The in vitro degradation assay indicates that ubiquitination at the N terminus partially contributes to the degradation of Smad1. Furthermore, we demonstrate that the ubiquitination level of pseudo-phosphorylated Smad1 by CHIP is stronger than that of wild-type Smad1 and can be strongly inhibited by a phosphorylated tail of Smad1, PIS(pS)V(pS). Third, our results indicate that Hsp70 facilitates CHIP-mediated poly-ubiquitination of Smad1 whereas it attenuates CHIP-meditated mono-ubiquitination of Smad1. Finally, consistent with the in vitro observation, we show that CHIP preferentially mediates the degradation of phospho-Smad1/5 in vivo. Taken together, these results provide us a hint that CHIP might preferentially regulate phosphorylated Smad1 and thus the BMP signaling.     

Synthesis of Optically Pure (1S, 4S, 5S)-2-Pinen-4-ol (cis-Verbenol) and Its Antipode,the Pheromone of Ips Bark Beetles

The enantiomers of cis-verbenol (4a and 4a′) were first synthesized in optically pure state. (1S, 4S, 5S)-2-Pinen-4-ol (4a′) was dextrorotatory in acetone or in methanol but it was levorotatory in chloroform; cis-verbenols are indistinguishable by a prefix (+) or (?). The designation of the Ips pheromone as (+)-cis-verbenol is therefore ambiguous and it should be called as (1S, 4S, 5S)-2-pinen-4-ol (4a′) or (S)-cis-verbenol.     

Elucidating the Mechanism of Substrate Recognition by the Bacterial Hsp90 Molecular Chaperone

Hsp90 is a conformationally dynamic molecular chaperone known to promote the folding and activation of a broad array of protein substrates (“clients”). Hsp90 is believed to preferentially interact with partially folded substrates, and it has been hypothesized that the chaperone can significantly alter substrate structure as a mechanism to alter the substrate functional state. However, critically testing the mechanism of substrate recognition and remodeling by Hsp90 has been challenging. Using a partially folded protein as a model system, we find that the bacterial Hsp90 adapts its conformation to the substrate, forming a binding site that spans the middle and C-terminal domains of the chaperone. Cross-linking and NMR measurements indicate that Hsp90 binds to a large partially folded region of the substrate and significantly alters both its local and long-range structure. These findings implicate Hsp90's conformational dynamics in its ability to bind and remodel partially folded proteins. Moreover, native-state hydrogen exchange indicates that Hsp90 can also interact with partially folded states only transiently populated from within a thermodynamically stable, native-state ensemble. These results suggest a general mechanism by which Hsp90 can recognize and remodel native proteins by binding and remodeling partially folded states that are transiently sampled from within the native ensemble.     

Comparison of gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods to quantify alpha-tocopherol and alpha-tocopherolquinone levels in human plasma.

Two mass spectrometric methods were established for the quantitative analyses of alpha-tocopherol (TH) and its oxidation product alpha-tocopherolquinone (TQ) in human plasma. Both methods make use of isotopically labeled internal standards of different levels of deuteration (d3-TH and d6-TQ). Plasma (100 microl) was saponified in the presence of a mixture of antioxidants, and then TH and TQ were extracted with hexane. With the GC-MS method, the analytes were first converted into O-trimethylsilyl derivatives before analysis in the selective ion monitoring mode. The derivatization procedure led to the quantitative conversion of TQ into the O-trimethylsilyl derivative of tocopherolhydroquinone, giving rise to a more stable molecule with less fragmentation than for TQ. The increased stability of the molecule resulted in an enhanced contribution of the base peak to the total observed ions and therefore an increased sensitivity of the base peak for quantification. With the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, TH and TQ were detected by multiple reaction monitoring after positive electrospray ionization. The GC-MS and LC-MS/MS methods showed nearly the same accuracy (>95%) and the same within-day precisions, with less than 5 and 10% for TH and TQ, respectively. The between-day precision and the limit of quantification for TQ in plasma were better by LC-MS/MS (4%; 3 nM) than by GC-MS (21%; 10 nM). Analysis and method validation were carried out with plasma samples obtained from a male volunteer pre- and postexercise. Both techniques showed that the ratio of TQ/TH was elevated by 35% immediately after exercise and had returned to basal levels when measured 24 h later.     

The alpha-kinases TRPM6 and TRPM7, but not eEF-2 kinase, phosphorylate the assembly domain of myosin IIA, IIB and IIC

TRPM6 and TRPM7 encode channel-kinases. While these channels share electrophysiological properties and cellular functions, TRPM6 and TRPM7 are non-redundant genes raising the possibility that the kinases have distinct substrates. Here, we demonstrate that TRPM6 and TRPM7 phosphorylate the assembly domain of myosin IIA, IIB and IIC on identical residues. Whereas phosphorylation of myosin IIA is restricted to the coiled-coil domain, TRPM6 and TRPM7 also phosphorylate the non-helical tails of myosin IIB and IIC. TRPM7 does not phosphorylate eukaryotic elongation factor-2 (eEF-2) and myosin II is a poor substrate for eEF-2 kinase. In conclusion, TRPM6 and TRPM7 share exogenous substrates among themselves but not with functionally distant alpha-kinases. STRUCTURED SUMMARY: