SPECC1L binds the myosin phosphatase complex MYPT1/PP1ß and can regulate its distribution between microtubules and filamentous actin.

The subcellular localization, activity , and substrate specificity of the serine/threonine protein phosphatase 1 catalytic subunit (PP1cat) is mediated through its dynamic association with regulatory subunits in holoenzyme complexes. While some functional overlap is observed for the three human PP1cat isoforms, they also show distinct targeting based on relative preferences for specific regulatory subunits. A well-known example is the preferential association of MYPT1 with PP1ß in the myosin phosphatase complex. In smooth muscle, MYPT1/PP1ß counteracts the muscle contraction induced by phosphorylation of the light chains of myosin by the myosin light chain kinase. This phosphatase complex is also found in nonmuscle cells, where it is targeted to both myosin and nonmyosin substrates and contributes to regulation of the balance of cytoskeletal structure and motility during cell migration and division. Although it remains unclear how MYPT1/PP1ß traffics between microtubule- and actin-associated substrates, our identification of the microtubule- and actin-binding protein SPECC1L in both the PP1ß and MYPT1 interactomes suggests that it is the missing link. Our validation of their association using coimmunoprecipitation and proximity biotinylation assays, together with the strong overlap that we observed for the SPECC1L and MYPT1 interactomes, confirmed that they exist in a stable complex in the cell. We further showed that SPECC1L binds MYPT1 directly and that it can impact the balance of the distribution of the MYPT1/PP1ß complex between the microtubule and filamentous actin networks.

Identification of Cdk1-LATS-Pin1 as a Novel Signaling Axis in Anti-tubulin Drug Response of Cancer Cells.

The Hippo pathway is a signaling cascade that plays important roles in organ size control, tumorigenesis, metastasis, stress response, stem cell differentiation, and renewal during development and tissue homeostasis and mechanotransduction. Recently, it has been observed that loss of the Hippo pathway core component LATS (large tumor suppressor) or overexpression of its downstream targets YAP and its paralog TAZ causes resistance of cancer cells to anti-tubulin drugs. However, YAP and TAZ mediates anti-tubulin drug-induced apoptosis independent of its upstream regulator LATS and the Hippo pathway. Thus, the underlying molecular mechanism of how LATS is involved in the anti-tubulin drug response remains unknown. Proteomic approaches, SILAC and BioID, were used to identify the isomerase Pin1 as a novel LATS-interacting protein after anti-tubulin drug treatment. Treatment with anti-tubulin drugs activated cyclin-dependent kinase 1 (CDK1), which phosphorylates LATS2 at five S/T-P motifs that functionally interact with the WW domain of Pin1 and inhibit its antiapoptotic function. Thus, these data identify Cdk1 and Pin1 as a novel upstream regulator and downstream mediator, respectively, of LATS in antitubulin drug response. Further studies on this novel Cdk1-LATS-Pin1 signaling axis will be important for understanding the molecular mechanisms of drug resistance and will provide useful information for targeting of this pathway in the future.Implications: This study provides new insight on the molecular mechanism of anti-tubulin drug resistance and suggests novel therapeutic targets for drug-resistant cancers. Mol Cancer Res; 16(6); 1035-45. ©2018 AACR.

Recent advances in large-scale protein interactome mapping.

Protein-protein interactions (PPIs) underlie most, if not all, cellular functions. The comprehensive mapping of these complex networks of stable and transient associations thus remains a key goal, both for systems biology-based initiatives (where it can be combined with other 'omics' data to gain a better understanding of functional pathways and networks) and for focused biological studies. Despite the significant challenges of such an undertaking, major strides have been made over the past few years. They include improvements in the computation prediction of PPIs and the literature curation of low-throughput studies of specific protein complexes, but also an increase in the deposition of high-quality data from non-biased high-throughput experimental PPI mapping strategies into publicly available databases.

Extracting, enriching, and identifying nuclear body sub-complexes using label-based quantitative mass spectrometry.

Determining the proteome of a nuclear body is a crucial step toward understanding its function; however, it is extremely challenging to obtain pure nuclear body preparations. Moreover, many nuclear proteins dynamically associate with multiple bodies and subnuclear compartments, confounding analysis. We have found that a more practical approach is to carry out affinity purification of nuclear body sub-complexes via the use of tagged nuclear-body-specific marker proteins. Here we describe in detail the method to identify new nuclear body protein sub-complexes through SILAC (stable isotope labeling by amino acids in culture)-based affinity purification followed by quantitative mass spectrometry.

Novel methods for studying multiprotein complexes in vivo.

The current consensus is that the majority of proteins act in concert in the cell, as homo- and heteromeric complexes of two or more proteins that carry out discrete biological functions. A wide range of genomic, proteomic, biochemical, structural and biophotonic techniques have been employed over the years to study the protein-protein interactions that define complexes, with the end goal of producing a spatiotemporal map of these modular functional units throughout the cell. Recent advances in the analysis of in vivo complexes have greatly improved structural, functional and temporal resolution, and this review highlights novel approaches ranging from proximity-dependent labeling and cross-linking/mass spectrometry through pulse-chase epitope labeling and targeted protein degradation.

ß-catenin is essential for efficient in vitro premyogenic mesoderm formation but can be partially compensated by retinoic acid signalling.

Previous studies have shown that P19 cells expressing a dominant negative ß-catenin mutant (ß-cat/EnR) cannot undergo myogenic differentiation in the presence or absence of muscle-inducing levels of retinoic acid (RA). While RA could upregulate premyogenic mesoderm expression, including Pax3/7 and Meox1, only Pax3/7 and Gli2 could be upregulated by RA in the presence of ß-cat/EnR. However, the use of a dominant negative construct that cannot be compensated by other factors is limiting due to the possibility of negative chromatin remodelling overriding compensatory mechanisms. In this study, we set out to determine if ß-catenin function is essential for myogenesis with and without RA, by creating P19 cells with reduced ß-catenin transcriptional activity using an shRNA approach, termed P19[shß-cat] cells. The loss of ß-catenin resulted in a reduction of skeletal myogenesis in the absence of RA as early as premyogenic mesoderm, with the loss of Pax3/7, Eya2, Six1, Meox1, Gli2, Foxc1/2, and Sox7 transcript levels. Chromatin immunoprecipitation identified an association of ß-catenin with the promoter region of the Sox7 gene. Differentiation of P19[shß-cat] cells in the presence of RA resulted in the upregulation or lack of repression of all of the precursor genes, on day 5 and/or 9, with the exception of Foxc2. However, expression of Sox7, Gli2, the myogenic regulatory factors and terminal differentiation markers remained inhibited on day 9 and overall skeletal myogenesis was reduced. Thus, ß-catenin is essential for in vitro formation of premyogenic mesoderm, leading to skeletal myogenesis. RA can at least partially compensate for the loss of ß-catenin in the expression of many myogenic precursor genes, but not for myoblast gene expression or overall myogenesis.

Skeletal myosin light chain kinase regulates skeletal myogenesis by phosphorylation of MEF2C.

The MEF2 factors regulate transcription during cardiac and skeletal myogenesis. MEF2 factors establish skeletal muscle commitment by amplifying and synergizing with MyoD. While phosphorylation is known to regulate MEF2 function, lineage-specific regulation is unknown. Here, we show that phosphorylation of MEF2C on T(80) by skeletal myosin light chain kinase (skMLCK) enhances skeletal and not cardiac myogenesis. A phosphorylation-deficient MEF2C mutant (MEFT80A) enhanced cardiac, but not skeletal myogenesis in P19 stem cells. Further, MEFT80A was deficient in recruitment of p300 to skeletal but not cardiac muscle promoters. In gain-of-function studies, skMLCK upregulated myogenic regulatory factor (MRF) expression, leading to enhanced skeletal myogenesis in P19 cells and more efficient myogenic conversion. In loss-of-function studies, MLCK was essential for efficient MRF expression and subsequent myogenesis in embryonic stem (ES) and P19 cells as well as for proper activation of quiescent satellite cells. Thus, skMLCK regulates MRF expression by controlling the MEF2C-dependent recruitment of histone acetyltransferases to skeletal muscle promoters. This work identifies the first kinase that regulates MyoD and Myf5 expression in ES or satellite cells.

MyoD directly up-regulates premyogenic mesoderm factors during induction of skeletal myogenesis in stem cells.

Gain- and loss-of-function experiments have illustrated that the family of myogenic regulatory factors is necessary and sufficient for the formation of skeletal muscle. Furthermore, MyoD required cellular aggregation to induce myogenesis in P19 embryonal carcinoma stem cells. To determine the mechanism by which stem cells can be directed into skeletal muscle, a time course of P19 cell differentiation was examined in the presence and absence of exogenous MyoD. By quantitative PCR, the first MyoD up-regulated transcripts were the premyogenic mesoderm factors Meox1, Pax7, Six1, and Eya2 on day 4 of differentiation. Subsequently, the myoblast markers myogenin, MEF2C, and Myf5 were up-regulated, leading to skeletal myogenesis. These results were corroborated by Western blot analysis, showing up-regulation of Pax3, Six1, and MEF2C proteins, prior to myogenin protein expression. To determine at what stage a dominant-negative MyoD/EnR mutant could inhibit myogenesis, stable cell lines were created and examined. Interestingly, the premyogenic mesoderm factors, Meox1, Pax3/7, Six1, Eya2, and Foxc1, were down-regulated, and as expected, skeletal myogenesis was abolished. Finally, to identify direct targets of MyoD in this system, chromatin immunoprecipitation experiments were performed. MyoD was observed associated with regulatory regions of Meox1, Pax3/7, Six1, Eya2, and myogenin genes. Taken together, MyoD directs stem cells into the skeletal muscle lineage by binding and activating the expression of premyogenic mesoderm genes, prior to activating myoblast genes.

Canonical Wnt signaling regulates Foxc1/2 expression in P19 cells.

FOXC1 and FOXC2 are forkhead/winged-helix transcription factors expressed in paraxial mesoderm and somites. Emphasizing the importance of FOXC1/2 during embryonic development, double-knockout mice lacking the alleles for both Foxc1 and Foxc2 failed to form segmented somites and undergo myogenesis. The present study aims to determine upstream factors that regulate Foxc1/2 expression during the differentiation of P19 cells into skeletal muscle. Previous work had shown that dominant-negative forms of beta-catenin, Gli2, and Meox1 could inhibit distinct stages of skeletal myogenesis in P19 cells. In the presence of a dominant-negative beta-catenin fusion protein, Foxc1/2 transcripts were not upregulated and neither were markers of somitogenesis/myogenesis, including Meox1, Pax3 and MyoD. Conversely, inhibition of GSK3 by LiCl or overexpression of activated beta-catenin in aggregated P19 cells resulted in enhancement of Foxc1/2 expression, indicating that FOX transcription may be under the control of Wnt signaling. Supporting this hypothesis, beta-catenin bound to conserved regions upstream of Foxc1 during P19 cell differentiation and drove transcription from this region in a promoter assay. In addition, ectopic expression of a dominant-negative Meox1 or Gli2 resulted in decreased Foxc1/2 transcript levels, correlating with inhibition of skeletal myogenesis. Overexpression of Gli2 was also sufficient to upregulate Foxc1/2 transcript levels and induce skeletal myogenesis. In summary, Foxc1/2 expression is dependent on a complex interplay from various signaling inputs from the Wnt and Shh pathways during early stages of in vitro skeletal myogenesis.

Retinoic acid enhances skeletal muscle progenitor formation and bypasses inhibition by bone morphogenetic protein 4 but not dominant negative beta-catenin.

BACKGROUND: Understanding stem cell differentiation is essential for the future design of cell therapies. While retinoic acid (RA) is the most potent small molecule enhancer of skeletal myogenesis in stem cells, the stage and mechanism of its function has not yet been elucidated. Further, the intersection of RA with other signalling pathways that stimulate or inhibit myogenesis (such as Wnt and BMP4, respectively) is unknown. Thus, the purpose of this study is to examine the molecular mechanisms by which RA enhances skeletal myogenesis and interacts with Wnt and BMP4 signalling during P19 or mouse embryonic stem (ES) cell differentiation. RESULTS: Treatment of P19 or mouse ES cells with low levels of RA led to an enhancement of skeletal myogenesis by upregulating the expression of the mesodermal marker, Wnt3a, the skeletal muscle progenitor factors Pax3 and Meox1, and the myogenic regulatory factors (MRFs) MyoD and myogenin. By chromatin immunoprecipitation, RA receptors (RARs) bound directly to regulatory regions in the Wnt3a, Pax3, and Meox1 genes and RA activated a beta-catenin-responsive promoter in aggregated P19 cells. In the presence of a dominant negative beta-catenin/engrailed repressor fusion protein, RA could not bypass the inhibition of skeletal myogenesis nor upregulate Meox1 or MyoD. Thus, RA functions both upstream and downstream of Wnt signalling. In contrast, it functions downstream of BMP4, as it abrogates BMP4 inhibition of myogenesis and Meox1, Pax3, and MyoD expression. Furthermore, RA downregulated BMP4 expression and upregulated the BMP4 inhibitor, Tob1. Finally, RA inhibited cardiomyogenesis but not in the presence of BMP4. CONCLUSION: RA can enhance skeletal myogenesis in stem cells at the muscle specification/progenitor stage by activating RARs bound directly to mesoderm and skeletal muscle progenitor genes, activating beta-catenin function and inhibiting bone morphogenetic protein (BMP) signalling. Thus, a signalling pathway can function at multiple levels to positively regulate a developmental program and can function by abrogating inhibitory pathways. Finally, since RA enhances skeletal muscle progenitor formation, it will be a valuable tool for designing future stem cell therapies.