FILIP1 CT pull-down
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive Plus
ORGANISM(S): Mus musculus
TISSUE(S): Skeletal Muscle Cell Line
DISEASE(S): Not Available
SUBMITTER: 
Friedel Drepper
LAB HEAD: Bettina Warscheid
PROVIDER: PXD008875 | Pride | 2022-06-06
REPOSITORIES: pride
Dataset's files
primary
| Action | |||
|---|---|---|---|
| QEplus018193.dat | Other | ||
| QEplus018193.mzid.gz | Mzid | ||
| QEplus018193.mzid_QEplus018193.MGF | Mzid | ||
| QEplus018193.raw | Raw | ||
| QEplus018195.dat | Other |
Items per page:
5
1 - 5 of 9
Publications
Phosphoproteomics identifies dual-site phosphorylation in an extended basophilic motif regulating FILIP1-mediated degradation of filamin-C.
Reimann Lena L Schwäble Anja N AN Fricke Anna L AL Mühlhäuser Wignand W D WWD Leber Yvonne Y Lohanadan Keerthika K Puchinger Martin G MG Schäuble Sascha S Faessler Erik E Wiese Heike H Reichenbach Christa C Knapp Bettina B Peikert Christian D CD Drepper Friedel F Hahn Udo U Kreutz Clemens C van der Ven Peter F M PFM Radziwill Gerald G Djinović-Carugo Kristina K Fürst Dieter O DO Warscheid Bettina B
Communications biology 20200522 1
The PI3K/Akt pathway promotes skeletal muscle growth and myogenic differentiation. Although its importance in skeletal muscle biology is well documented, many of its substrates remain to be identified. We here studied PI3K/Akt signaling in contracting skeletal muscle cells by quantitative phosphoproteomics. We identified the extended basophilic phosphosite motif RxRxxp[S/T]xxp[S/T] in various proteins including filamin-C (FLNc). Importantly, this extended motif, located in a unique insert in Ig- ...[more]
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Project description:Interaction between the cytoplasmic domain of GPIb? with its cytoskeletal binding partner, filamin, is a major determinant of platelet size, and deficiency of either protein results in macrothrombocytopenia. To clarify the mechanism by which GPIb?-filamin interactions regulate platelet production, we manipulated the expression levels of filamin and GPIb in cultured embryonic stem cells (ESCs) that were subsequently differentiated into platelets. Knocking down filamins A and B resulted in the production of ESC-derived proplatelets with abnormally large swellings and proplatelet shafts that generated giant platelets in culture. Large platelets could also be generated by overexpressing GPIb? in ESCs, or by overexpressing in vivo a transgene encoding a chimeric protein containing the cytoplasmic domain of GPIb?. To identify the mechanism by which the GPIb:filamin ratio regulates platelet size, we manipulated filamin and GPIb? levels in HEK293T cells and examined the effects of overexpressing either protein on their ability to traffic to the cell periphery. Accumulation of either protein within the endoplasmic reticulum resulted in trapping of the other. Taken together, these data demonstrate that coordinated expression of GPIb? and filamin is required for efficient trafficking of either protein to the cell surface, and for production of normal-sized platelets.
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Project description:Platelet microparticles are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and platelet microparticle levels. Yet little is known about the processes by which platelet microparticles are generated in vivo. We now characterize microparticles derived directly from megakaryocytes. Video microscopy of live mouse megakaryocytes demonstrated that microparticles form as submicron beads along the lengths of slender, unbranched micropodia. These microparticles are CD41(+), CD42b(+), and express surface phosphatidylserine. Megakaryocyte microparticle generation is resistant to inhibition of microtubule assembly, which is critical to platelet formation, and augmented by inhibition of actin polymerization. To determine whether circulating microparticles are derived primarily from activated platelets or megakaryocytes, we identified markers that distinguish between these 2 populations. CD62P and LAMP-1 were found only on mouse microparticles from activated platelets. In contrast, full-length filamin A was found in megakaryocyte-derived microparticles, but not microparticles from activated platelets. Circulating microparticles isolated from mice were CD62P(-), LAMP-1(-) and expressed full-length filamin A, indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62P(-) and expressed full-length filamin A. Cultured human megakaryocytes elaborated microparticles that were CD41(+), CD42b(+), and express surface phosphatidylserine. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet microparticles.
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Project description:Post translational protein modifications (PTMs) including small chemical groups and small proteins, belonging to the ubiquitin family, are essential for virtually all cellular processes. In addition to modification by a single PTM, proteins can be modified by a combination of different modifiers, which are able to influence each other. Since little is known about crosstalk between different ubiquitin family members, we developed an improved method enabling identification of co-modified proteins on a system-wide level using mass spectrometry. We focused on the role of crosstalk between SUMO and ubiquitin during proteasomal degradation. Using two complementary approaches, we identified 498 proteins to be significantly co-modified by SUMO and ubiquitin upon MG132 treatment. These targets included many enzymatic components of PTM machinery, involved in SUMOylation and ubiquitylation, but also phosphorylation, methylation and acetylation, revealing a highly complex interconnected network of crosstalk between different PTMs. In addition various other biological processes were found to be significantly enriched within the group of co-modified proteins, including transcription, DNA repair and the cell cycle. Interestingly, the latter group mostly consisted of proteins involved in mitosis, including a subset of chromosome segregation regulators. We hypothesize that group modification by SUMO-targeted ubiquitin ligases regulates the stability of the identified subset of mitotic proteins, which ensures proper chromosome segregation. The mitotic regulators KIF23 and MIS18BP1 were verified to be co-modified by SUMO and ubiquitin upon inhibition of the proteasome and subsequently identified as novel RNF4 targets. Both modifications on MIS18BP1 were observed to increase simultaneously during late mitosis, while the total protein level decreased immediately afterwards. These results confirm the regulation of MIS18BP1 via SUMO-ubiquitin crosstalk during mitosis. Combined, our work highlights extensive crosstalk between SUMO and ubiquitin, providing a resource for further unraveling of SUMO-ubiquitin crosstalk.
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Project description:Filaminopathy is a subtype of myofibrillar myopathy caused by mutations in FLNC, the gene encoding filamin C, and histologically characterized by pathologic accumulation of several proteins within skeletal muscle fibers. With the aim to get new insights in aggregate composition, we collected aggregates and control tissue from skeletal muscle biopsies of six myofibrillar myopathy patients harboring three different FLNC mutations by laser microdissection and analyzed the samples by a label-free mass spectrometry approach. A total of 390 proteins were identified, and 31 of those showed significantly higher spectral indices in aggregates compared with patient controls with a ratio >1.8. These proteins included filamin C, other known myofibrillar myopathy associated proteins, and a striking number of filamin C binding partners. Across the patients the patterns were extremely homogeneous. Xin actin-binding repeat containing protein 2, heat shock protein 27, nebulin-related-anchoring protein, and Rab35 could be verified as new filaminopathy biomarker candidates. In addition, further experiments identified heat shock protein 27 and Xin actin-binding repeat containing protein 2 as novel filamin C interaction partners and we could show that Xin actin-binding repeat containing protein 2 and the known interaction partner Xin actin-binding repeat containing protein 1 simultaneously associate with filamin C. Ten proteins showed significant lower spectral indices in aggregate samples compared with patient controls (ratio <0.56) including M-band proteins myomesin-1 and myomesin-2. Proteomic findings were consistent with previous and novel immunolocalization data. Our findings suggest that aggregates in filaminopathy have a largely organized structure of proteins also interacting under physiological conditions. Different filamin C mutations seem to lead to almost identical aggregate compositions. The finding that filamin C was detected as highly abundant protein in aggregates in filaminopathy indicates that our proteomic approach may be suitable to identify new candidate genes among the many MFM patients with so far unknown mutation.
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Project description:Filamin C is mainly expressed in striated muscle cells where it localizes to Z-discs, myotendinous junctions and intercalated discs. Recent studies have revealed numerous mutations in the FLNC gene causing familiar and sporadic myopathies and cardiomyopathies with marked clinical variability. The most frequent myopathic mutation, p.W2710X, which is associated with myofibrillar myopathy, deletes the carboxy-terminal 16 amino acids from filamin C and abolishes the dimerization property of Ig-like domain 24. We previously characterized "knock-in" mice heterozygous for this mutation (p.W2711X), and have now investigated homozygous mice using protein and mRNA expression analyses, mass spectrometry, and extensive immunolocalization and ultrastructural studies. Although the latter mice display a relatively mild myopathy under normal conditions, our analyses identified major mechanisms causing the pathophysiology of this disease: (i) the expression level of filamin C protein is drastically reduced; (ii) mutant filamin C is relocalized from Z-discs to particularly mechanically strained parts of muscle cells, i.e. myotendinous junctions and myofibrillar lesions; (iii) the number of lesions is greatly increased and these lesions lack BAG3; (iv) the expression of HSPB7 is almost completely abolished. These findings indicate grave disturbances of BAG3-dependent and -independent autophagy pathways that are required for efficient lesion repair. In addition, our studies reveal general mechanisms of lesion formation and demonstrate that defective filamin C dimerization via its carboxy-terminal domain does not disturb assembly and basic function of myofibrils. An alternative dimerization site might compensate for that loss. Since filamins function as stress sensors, our data further substantiate that filamin C is important for mechanosensing in the context of Z‑disc stabilization and maintenance.
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