Component Of Filaments Research Articles

Nuclear Matrix Proteome Analysis of Drosophila melanogaster

The nucleus is a highly structured organelle and contains many functional compartments. Although the structural basis for this complex spatial organization of compartments is unknown, a major component of this organization is likely to be the non-chromatin scaffolding called nuclear matrix (NuMat). Experimental evidence over the past decades indicates that most of the nuclear functions are at least transiently associated with the NuMat, although the components of NuMat itself are poorly known. Here, we report NuMat proteome analysis from Drosophila melanogaster embryos and discuss its links with nuclear architecture and functions. In the NuMat proteome, we found structural proteins, chaperones, DNA/RNA-binding proteins, chromatin remodeling and transcription factors. This complexity of NuMat proteome is an indicator of its structural and functional significance. Comparison of the two-dimensional profile of NuMat proteome from different developmental stages of Drosophila embryos showed that less than half of the NuMat proteome is constant, and the rest of the proteins are stage-specific dynamic components. These NuMat dynamics suggest a possible functional link between NuMat and embryonic development. Finally, we also showed that a subset of NuMat proteins remains associated with the mitotic chromosomes, implicating their role in mitosis and possibly the epigenetic cellular memory. NuMat proteome analysis provides tools and opens up ways to understand nuclear organization and function.

Characterization and functional analysis of seven flagellin genes in Rhizobium leguminosarum bv. viciae. Characterization of R. leguminosarum flagellins

BackgroundRhizobium leguminosarum bv. viciae establishes symbiotic nitrogen fixing partnerships with plant species belonging to the Tribe Vicieae, which includes the genera Vicia, Lathyrus, Pisum and Lens. Motility and chemotaxis are important in the ecology of R. leguminosarum to provide a competitive advantage during the early steps of nodulation, but the mechanisms of motility and flagellar assembly remain poorly studied. This paper addresses the role of the seven flagellin genes in producing a functional flagellum.ResultsR. leguminosarum strains 3841 and VF39SM have seven flagellin genes (flaA, flaB, flaC, flaD, flaE, flaH, and flaG), which are transcribed separately. The predicted flagellins of 3841 are highly similar or identical to the corresponding flagellins in VF39SM. flaA, flaB, flaC, and flaD are in tandem array and are located in the main flagellar gene cluster. flaH and flaG are located outside of the flagellar/motility region while flaE is plasmid-borne. Five flagellin subunits (FlaA, FlaB, FlaC, FlaE, and FlaG) are highly similar to each other, whereas FlaD and FlaH are more distantly related. All flagellins exhibit conserved amino acid residues at the N- and C-terminal ends and are variable in the central regions. Strain 3841 has 1-3 plain subpolar flagella while strain VF39SM exhibits 4-7 plain peritrichous flagella. Three flagellins (FlaA/B/C) and five flagellins (FlaA/B/C/E/G) were detected by mass spectrometry in the flagellar filaments of strains 3841 and VF39SM, respectively. Mutation of flaA resulted in non-motile VF39SM and extremely reduced motility in 3841. Individual mutations of flaB and flaC resulted in shorter flagellar filaments and consequently reduced swimming and swarming motility for both strains. Mutant VF39SM strains carrying individual mutations in flaD, flaE, flaH, and flaG were not significantly affected in motility and filament morphology. The flagellar filament and the motility of 3841 strains with mutations in flaD and flaG were not significantly affected while flaE and flaH mutants exhibited shortened filaments and reduced swimming motility.ConclusionThe results obtained from this study demonstrate that FlaA, FlaB, and FlaC are major components of the flagellar filament while FlaD and FlaG are minor components for R. leguminosarum strains 3841 and VF39SM. We also observed differences between the two strains, wherein FlaE and FlaH appear to be minor components of the flagellar filaments in VF39SM but these flagellin subunits may play more important roles in 3841. This paper also demonstrates that the flagellins of 3841 and VF39SM are possibly glycosylated.

Trypanosoma cruzi: Identification of DNA targets of the nuclear periphery coiled-coil protein TcNUP-1

The nuclear lamina is a structure that lines the inner nuclear membrane. In metazoans, lamins are the primary structural components of the nuclear lamina and are involved in several processes. Eukaryotes that lack lamins have distinct proteins with homologous functions. Some years ago, a coiled-coil protein in Trypanosoma brucei, NUP-1, was identified as the major filamentous component of its nuclear lamina. However, its precise role has not been determined. We characterized a homologous protein in Trypanosoma cruzi, TcNUP-1, and identified its in vivo DNA binding sites using a chromatin immunoprecipitation assay. We demonstrate for the first time that TcNUP-1 associates with chromosomal regions containing large non-tandem arrays of genes encoding surface proteins. We therefore suggest that TcNUP-1 is a structural protein that plays an essential role in nuclear organization by anchoring T. cruzi chromosomes to the nuclear envelope.

Tyrosine phosphorylation of tau accompanies disease progression in transgenic mouse models of tauopathy

Tau protein is a prominent component of paired helical filaments in Alzheimer's disease (AD) and other tauopathies. While the abnormal phosphorylation of tau on serine and threonine has been well established in the disease process, its phosphorylation on tyrosine has only recently been described. We previously showed that the Src family non-receptor tyrosine kinases (SFKs) Fyn and Src phosphorylate tau on Tyr18 and that phospho-Tyr18-tau was present in AD brain. In this study, we have investigated the appearance of phospho-Tyr18-tau, activated SFK and proliferating cell nuclear antigen (PCNA) during disease progression in a mouse model of human tauopathy. We have used JNPL3, which expresses human tau with P301L mutation, and antibodies specific for phospho-Tyr18-tau (9G3), ser/thr phosphorylated tau (AT8), activated SFK and PCNA. Antibody staining was viewed by either epifluorescence or confocal microscopy. Phospho-Tyr18-tau appeared concurrently with AT8-reactive tau as early as 4 months in JNPL3. Some 9G3-positive cells also contained activated SFKs and PCNA. We also investigated the triple transgenic mouse model of AD and found that unlike the JNPL3 model, the appearance of 9G3 reactivity did not coincide with AT8 in the hippocampus, suggesting that the presence of APP/presenilin influences tau phosphorylation. Also, Thioflavin S-positive plaques were 9G3-negative, suggesting that phospho-Tyr18-tau is absent from the dystrophic neurites of the mouse triple transgenic brain. Our results provide evidence for the association of tyrosine-phosphorylated tau with mechanisms of neuropathogenesis and indicate that SFK activation and cell cycle activation are also involved in JNPL3.

Nebulin regulates actin filament lengths by a stabilization mechanism

Efficient muscle contraction requires regulation of actin filament lengths. In one highly cited model, the giant protein nebulin has been proposed to function as a molecular ruler specifying filament lengths. We directly challenged this hypothesis by constructing a unique, small version of nebulin (mini-nebulin). When endogenous nebulin was replaced with mini-nebulin in skeletal myocytes, thin filaments extended beyond the end of mini-nebulin, an observation which is inconsistent with a strict ruler function. However, under conditions that promote actin filament depolymerization, filaments associated with mini-nebulin were remarkably maintained at lengths either matching or longer than mini-nebulin. This indicates that mini-nebulin is able to stabilize portions of the filament it has no contact with. Knockdown of nebulin also resulted in more dynamic populations of thin filament components, whereas expression of mini-nebulin decreased the dynamics at both filament ends (i.e., recovered loss of endogenous nebulin). Thus, nebulin regulates thin filament architecture by a mechanism that includes stabilizing the filaments and preventing actin depolymerization.

Multiple Functional Domains of the Yeast l,3-β-Glucan Synthase Subunit Fks1p Revealed by Quantitative Phenotypic Analysis of Temperature-Sensitive Mutants

The main filamentous structural component of the cell wall of the yeast Saccharomyces cerevisiae is 1,3-beta-glucan, which is synthesized by a plasma membrane-localized enzyme called 1,3-beta-glucan synthase (GS). Here we analyzed the quantitative cell morphology and biochemical properties of 10 different temperature-sensitive mutants of FKS1, a putative catalytic subunit of GS. To untangle their pleiotropic phenotypes, the mutants were classified into three functional groups. In the first group, mutants fail to synthesize 1,3-beta-glucan at the proper subcellular location, although GS activity is normal in vitro. In the second group, mutants have normal 1,3-beta-glucan content but are defective in polarized growth and endocytosis. In the third group, mutations in the putative catalytic domain of Fks1p result in a loss of the catalytic activity of GS. The differences among the three groups suggest that Fks1p consists of multiple domains that are required for cell wall construction and cellular morphogenesis.

Effects of Tau on the activity of triose phosphate isomerase (TPI) in brain cells

Tau is a main component of the aberrant paired helical filaments (PHF) found in Alzheimer's disease (AD). It has also been reported to enhance oxidative stress, which is a major factor in the pathogenesis of neurodegenerative diseases. However, protective functions of Tau have recently been reported, including antagonizing apoptosis, in addition to its role in stabilizing microtubules. In this study, the interaction between Tau and triose phosphate isomerase (TPI) in a normal, nondisease state as well as in a neurodegeneration state was examined and demonstrated for the first time. More importantly, we also showed that Tau protects TPI against oxidative damage. An oxidative stress-induced decrease in the activity of TPI was attenuated in Tau-overexpressing cells, indicating that Tau protects TPI against oxidative damage. By contrast, the activity of TPI was decreased in Tau-transgenic (Tg) mice compared to non-Tg (NTg) mice even though protein levels were not changed in both groups. Some TPIs were found on the PHF in Tg mice, which explains the decrease in the activity of TPI. Taken together, we concluded that while Tau binds and protects TPI in normal cells, and conversely, the formation of PHF induced by Tau phosphorylation trap some TPI and trigger the functional loss of TPI in the development of neurodegenerative diseases. Our results provide new insights into understanding the in-depth involvement of Tau in the development of neurodegenerative disorders.

Phosphorylation motifs in the nonhelical domains of myosin heavy chain and paramyosin may negatively regulate assembly in Caenorhabditis elegans striated muscle

We are interested in mechanisms that establish and maintain the highly ordered contractile apparatus of striated muscle. The homologous proteins myosin and paramyosin are the major structural components of thick filaments in invertebrate animals. In Caenorhabditis elegans, both proteins contain a homologous, small nonhelical domain that is known to be phosphorylated in paramyosin. In this report, we show that a proposed phosphorylation motif (S_S_A), which is present in several copies in the nonhelical regions of both myosin and paramyosin, is highly conserved among nematodes. We used in vivo assays to examine the assembly properties of proteins in which one or more motifs were targeted by point mutagenesis or deletion. In all cases, expression of mutant proteins improved the phenotype of the corresponding null mutant animals, but produced variable structural defects, including birefringent aggregates in adults and abnormal localization in embryos. Point mutation, but not deletion, of the myosin A nonhelical tailpiece produced ectopic structures that appeared as masses of jumbled filaments by TEM. Antibody labeling showed that aggregates of either mutant protein did not recruit the endogenous version of the other. Analysis of mutant embryos lacking either paramyosin or myosin A (the essential isoform at the thick filament center) indicated that both wild-type proteins can independently localize and initiate assembly, although the structures produced are abnormal. Our results suggest that muscle cells actively restrict myosin and paramyosin assembly through phosphorylation of the S_S_A motifs and that each protein is regulated independently.

Relationship Between Tau Pathology and Neuroinflammation in Alzheimer's Disease

Alzheimer's disease is a chronic, age-related neurodegenerative disorder. Neurofibrillary tangles are among the pathological hallmarks of Alzheimer's disease. Neurofibrillary tangles consist of abnormal protein fibers known as paired helical filaments. The accumulation of paired helical filaments is one of the most characteristic cellular changes in Alzheimer's disease. Tau protein, a microtubule-associated protein, is the major component of paired helical filaments. Tau in paired helical filaments is hyperphosphorylated, truncated, and aggregated. What triggers the formation of paired helical filaments is not known, but neuroinflammation could play a role. Neuroinflammation is an active process detectable in the earliest stages of Alzheimer's disease. The neuronal toxicity associated with inflammation makes it a potential risk factor in the pathogenesis of Alzheimer's disease. Determining the sequence of events that lead to this devastating disease has become one of the most important goals for the prevention and treatment of Alzheimer's disease. In this review, we focus on the pathological properties of tau thought to play a role in neurofibrillary tangle formation and summarize how central nervous system inflammation might be a critical contributor to the pathology of Alzheimer's disease. A better understanding of the mechanisms that cause neurofibrillary tangle formation is of clinical importance for developing therapeutic strategies to prevent and treat Alzheimer's disease. One of the major challenges facing us is singling out neuroinflammation as a therapeutic target for the prevention of Alzheimer's disease neurodegeneration. The challenge is developing therapeutic strategies that prevent neurotoxicity linked to inflammation without compromising its neuroprotective role.

Caenorhabditis elegans unc-82 encodes a serine/threonine kinase important for myosin filament organization in muscle during growth.

Mutations in the unc-82 locus of Caenorhabditis elegans were previously identified by screening for disrupted muscle cytoskeleton in otherwise apparently normal mutagenized animals. Here we demonstrate that the locus encodes a serine/threonine kinase orthologous to human ARK5/SNARK (NUAK1/NUAK2) and related to the PAR-1 and SNF1/AMP-Activated kinase (AMPK) families. The predicted 1600-amino-acid polypeptide contains an N-terminal catalytic domain and noncomplex repetitive sequence in the remainder of the molecule. Phenotypic analyses indicate that unc-82 is required for maintaining the organization of myosin filaments and internal components of the M-line during cell-shape changes. Mutants exhibit normal patterning of cytoskeletal elements during early embryogenesis. Defects in localization of thick filament and M-line components arise during embryonic elongation and become progressively more severe as development proceeds. The phenotype is independent of contractile activity, consistent with unc-82 mutations preventing proper cytoskeletal reorganization during growth, rather than undermining structural integrity of the M-line. This is the first report establishing a role for the UNC-82/ARK5/SNARK kinases in normal development. We propose that activation of UNC-82 kinase during cell elongation regulates thick filament attachment or growth, perhaps through phosphorylation of myosin and paramyosin. We speculate that regulation of myosin is an ancestral characteristic of kinases in this region of the kinome.

Study of flagella-mediated interactions between plants and Pseudomonas syringae

The phytopathogenic bacterium P. syringae can be classified into more than 50 pathovars on the basis of their host plant species and the disease symptoms they cause. They enter apoplastic spaces of host plants via natural openings such as wounds and stomata in leaves, stems, and fruits, then multiply and cause leaf spots, necrosis, wilt and stem canker. Each P. syringae pathovar has its own host species. For example, P. syringae pv. tabaci (Pta) 6605 is the causal agent of wild fire disease in tobacco, but does not cause disease in nonhost plants such as tomato and Arabidopsis thaliana. In Gram-negative phytopathogenic bacteria, a hrp (hypersensitive reaction and pathogenicity) gene cluster is required to cause the hypersensitive reaction (HR) on nonhost plants and pathogenicity on host plants. The hrp gene cluster encodes components of the Hrp type III secretion apparatus, regulatory proteins for hrp gene expression and the harpin protein, a proteinaceous elicitor effective on nonhost plants. Harpin is encoded by hrpZ in the hrp gene cluster. We have investigated the hrpZ gene in several pathovars of P. syringae and revealed that hrpZ in strain Pta6605 has a deleterious internal deletion and frame shift, and thus does not produce active elicitor. Harpin is thought to be a major elicitor of HR in nonhost plants, and we predicted that Pta6605 has a different elicitor because Pta6605 does induce typical HR on nonhost tomato plants. By screening for active elicitor molecules in the culture supernatant of Pta6605, we identified the flagellin protein, a major component of the flagellar filament, as a strong elicitor of hypersensitive cell death in tomato.

Structural and Functional Diversity of Novel Coronin 1C (CRN2) Isoforms in Muscle

Coronin 1C (synonyms: coronin-3, CRN2), a WD40 repeat-containing protein involved in cellular actin dynamics, is ubiquitously expressed in human tissues. Here, we report on the identification and functional characterization of two novel coronin 1C isoforms, referred to as CRN2i2 and CRN2i3, which also associate with F-actin. Analyses of the coronin 1C gene disclosed a single promoter containing binding sites for myogenic regulatory factors and an alternative first exon 1b present in intron 1, which give rise to the novel isoforms. Chromatin immunoprecipitation studies demonstrate MyoD binding to a region of the CRN2 gene, which contains a highly conserved E-box element in exon 1a. Gel-filtration assays suggest that the largest isoform 3 exists as a monomer, in contrast to isoform 1 and isoform 2 appearing as trimers. CRN2i3, which can be induced by MyoD, is exclusively expressed in well-differentiated myoblasts as well as in mature skeletal muscle tissue. In human skeletal muscle, CRN2i3 is a novel component of postsynaptic neuromuscular junctions and thin filaments of myofibrils. Together, our findings postulate a role for CRN2 isoforms in the structural and functional organization of F-actin in highly ordered protein complexes.

Stage-dependent transcriptional changes and characterization of paramyosin of the bovine lungworm Dictyocaulus viviparus

The bovine lungworm Dictyocaulus viviparus is of major economic importance in cattle farming in the temperate zones. The invertebrate protein paramyosin is one of the main components of muscle thick filaments but can also exhibit immunomodulatory functions. It represents a promising vaccine candidate in parasitic helminths. In this study, D. viviparus paramyosin (DvPmy) was characterized on the transcriptional as well as genomic level. The identified genomic sequence comprises 19 introns compared to only 10 introns in the Caenorhabditis elegans orthologue. Quantitative real time PCR transcriptional analysis revealed paramyosin transcription throughout the whole parasite's life cycle with the highest transcription rate in the agile moving first-stage larvae and the lowest in motionless hypobiosis induced third stage larvae. Recombinantly expressed DvPmy was found to bind collagen and IgG. Thereby the present study is the first showing that nematode paramyosin has the capability for immunomodulation and thus may be involved in host immune defence.

During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation

During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation

Thiolutin inhibits endothelial cell adhesion by perturbing Hsp27 interactions with components of the actin and intermediate filament cytoskeleton

Thiolutin is a dithiole synthesized by Streptomyces sp. that inhibits endothelial cell adhesion and tumor growth. We show here that thiolutin potently inhibits developmental angiogenesis in zebrafish and vascular outgrowth from tissue explants in 3D cultures. Thiolutin is a potent and selective inhibitor of endothelial cell adhesion accompanied by rapid induction of HSPB1 (Hsp27) phosphorylation. The inhibitory effects of thiolutin on endothelial cell adhesion are transient, potentially due to a compensatory increase in Hsp27 protein levels. Accordingly, heat shock induction of Hsp27 limits the anti-adhesive activity of thiolutin. Thiolutin treatment results in loss of actin stress fibers, increased cortical actin as cells retract, and decreased cellular F-actin. Mass spectrometric analysis of Hsp27 binding partners following immunoaffinity purification identified several regulatory components of the actin cytoskeleton that associate with Hsp27 in a thiolutin-sensitive manner including several components of the Arp2/3 complex. Among these, ArpC1a is a direct binding partner of Hsp27. Thiolutin treatment induces peripheral localization of phosphorylated Hsp27 and Arp2/3. Hsp27 also associates with the intermediate filament components vimentin and nestin. Thiolutin treatment specifically ablates Hsp27 interaction with nestin and collapses nestin filaments. These results provide new mechanistic insights into regulation of cell adhesion and cytoskeletal dynamics by Hsp27.

Ala Scanning of the Inhibitory Region of Cardiac Troponin I

In skeletal and cardiac muscles, troponin (Tn), which resides on the thin filament, senses a change in intracellular Ca(2+) concentration. Tn is composed of TnC, TnI, and TnT. Ca(2+) binding to the regulatory domain of TnC removes the inhibitory effect by TnI on the contraction. The inhibitory region of cardiac TnI spans from residue 138 to 149. Upon Ca(2+) activation, the inhibitory region is believed to be released from actin, thus triggering actin-activation of myosin ATPase. In this study, we created a series of Ala-substitution mutants of cTnI to delineate the functional contribution of each amino acid in the inhibitory region to myofilament regulation. We found that most of the point mutations in the inhibitory region reduced the ATPase activity in the presence of Ca(2+), which suggests the same region also acts as an activator of the ATPase. The thin filaments can also be activated by strong myosin head (S1)-actin interactions. The binding of N-ethylmaleimide-treated myosin subfragment 1 (NEM-S1) to actin filaments mimics such strong interactions. Interestingly, in the absence of Ca(2+) NEM-S1-induced activation of S1 ATPase was significantly less with the thin filaments containing TnI(T144A) than that with the wild-type TnI. However, in the presence of Ca(2+), there was little difference in the activation of ATPase activity between these preparations.

During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation

Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1.

Regulatory mechanism of smooth muscle contraction studied with gelsolin-treated strips of taenia caeci in guinea pig

The potential roles of the regulatory proteins actin, tropomyosin (Tm), and caldesmon (CaD), i.e., the components of the thin filament, in smooth muscle have been extensively studied in several types of smooth muscles. However, controversy remains on the putative physiological significance of these proteins. In this study, we intended to determine the functional roles of Tm and CaD in the regulation of smooth muscle contraction by using a reconstitution system of the thin filaments. At appropriate conditions, the thin (actin) filaments within skinned smooth muscle strips of taenia caeci in guinea pigs could be selectively removed by an actin-severing protein, gelsolin, without irreversible damage to the contractile apparatus, and then the thin filaments were reconstituted with purified components of thin filaments, i.e., actin, Tm, and CaD. We found that the structural remodeling of actin filaments or thin filaments was functionally linked to the Ca(2+)-induced force development and reduction in muscle cross-sectional area (CSA). That is, after the reconstitution of the gelsolin-treated skinned smooth muscle strips with pure actin, the Ca(2+)-dependent force development was partially restored, but the Ca(2+)-induced reduction in CSA occurred once. In contrast, the reconstitution with actin, followed by Tm and CaD, restored not only the force generation but also both its Ca(2+) sensitivity and the reversible Ca(2+)-dependent reduction in CSA. We confirmed that both removal of the thin filaments by gelsolin treatment and reconstitution of the actin (thin) filaments with Tm and CaD caused no significant changes in the level of myosin regulatory light chain phosphorylation. We thus conclude that Tm and CaD are necessary for the full regulation of smooth muscle contraction in addition to the other regulatory systems, including the myosin-linked one.

The nuclear pore component Nup358 promotes transportin-dependent nuclear import

Nup358 (also known as RanBP2), a component of the cytoplasmic filaments of the nuclear pore complex, has been implicated in various nucleocytoplasmic transport pathways. Here, we identify Nup358 as an important factor for transportin-mediated nuclear import. Depletion of Nup358 resulted in a strong inhibition of nuclear import of the human immunodeficiency virus type 1 (HIV-1) Rev protein. HIV-1 Rev is an RNA-binding protein that is required for CRM1 (also known as exportin 1)-dependent nuclear export of unspliced or partially spliced viral RNA. We show that transportin is the major nuclear import receptor for HIV-1 Rev in HeLa cells. Overexpression of transportin strongly promoted nuclear import of HIV-1 Rev in Nup358-depleted cells, indicating that the import receptor becomes rate-limiting under these conditions. Importantly, the import rate of other transportin-dependent proteins was also significantly reduced in Nup358-depleted cells. Our data therefore suggest a general role for Nup358 in transportin-mediated nuclear import.

Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale

AMP-activated protein kinase (AMPK) is responsible for sensing of the cell’s energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades. Mutations in the gene encoding the γ2 regulatory subunit have been shown to cause hypertrophic cardiomyopathy (HCM) with conduction abnormalities. As part of a study to examine the role of AMPK in the heart, we tested whether specific domains of the thick filament component cardiac myosin binding protein-C (cMyBP-C) were good in vitro AMPK substrates. The commercially available pET28a expression vector was used to generate a recombinant form of the cMyBP-C C8 domain as a fusion protein with a hexahistidine tag. In vitro phosphorylation with activated kinase showed that the purified fusion protein was a good AMPK substrate, phosphorylated at a similar rate to the control SAMS peptide and with phosphate incorporation specifically in serine residues. However, subsequent analysis of alanine replacement mutants and thrombin digestion revealed that the strong AMPK phosphorylation site was contained within the thrombin cleavage sequence encoded by the vector. As this sequence is common to many commercial pET vectors, caution is advised in the mapping of AMPK phosphorylation sites when this sequence is present.