Journal: The Journal of Cell Biology

Article Title: Formin follows function: a muscle-specific isoform of FHOD3 is regulated by CK2 phosphorylation and promotes myofibril maintenance

doi: 10.1083/jcb.201005060

Figure Lengend Snippet: FHOD3 contains a novel alternative exon at the end of its FH2 domain. (A) Domain layout of the human FHOD3 splice isoform expressing the T(D/E) 5 XE exon (exon 26 in humans and exon 25 in rodents; Fig. S1 ). GBD, GTPase-binding domain; DID, diaphanous inhibitory domain; FH1/FH2, formin homology domains 1 and 2. (B) The alternative T(D/E) 5 XE exon of FHOD3 encodes for an evolutionarily conserved stretch of eight mainly acidic amino acids that introduce CK2 consensus sites. Lowercase letters are intronic, and capital letters represent exonic sequences. Splice acceptor and donor sites are evolutionarily conserved in all seven orthologues, with the intronic bases AG on the 5′ end and GT on the 3′ end. (C) GST fusion proteins of the FHOD3-FH2 domain containing (FH2L) or excluding (FH2S) the alternative T(D/E) 5 XE exon, as well as threonine to alanine mutants of the predicted target threonines (T1474, T1476, or both), and the FHOD1-FH2 domain were tested for CK2 in vitro phosphorylation. FH2L, but not FH2S or FHOD1-FH2, was phosphorylated by CK2. Mutation of T1474 or T1476 to alanine reduced phosphorylation slightly or greatly, whereas mutation of both residues completely abolished the phosphorylation. mM, monoclonal mouse. (D) A similar phosphorylation pattern to that seen in vitro could also be demonstrated in COS-1 cells transfected with different GFP-tagged FHOD3 constructs and was abolished by treatment with 10 µM of the CK2 inhibitor DMAT for 24 h before lysis. The signal for GFP is shown as a loading control below. (E) Schematic view of the primer sets used for the RT-PCRs. (F) RT-PCR to test for tissue-specific expression of the alternative T(D/E) 5 XE exon. The amount of template mouse cDNAs was normalized against glycerine aldehyde 3-phosphate dehydrogenase (GAPDH). The specificity of the T(D/E) 5 XE exon for striated muscle can either be seen with primer set E25-27 or, because of the difference in the electrophoretic mobility, with primer set E24-27. The top line points out the 503-bp fragment, and the bottom line indicates the 479-bp fragment, which are also indicated by the arrows. TA, tibialis anterior.

Article Snippet: For in cyto experiments, COS-1 cells or NRCs were grown for 24 h and then treated with 10 μM of the CK2 Inhibitor DMAT (InSolution; Merck Chemicals) for 24 h before cell lysis or fixation and antibody staining as described earlier.

Techniques: Expressing, Binding Assay, Introduce, In Vitro, Phospho-proteomics, Mutagenesis, Transfection, Construct, Lysis, Control, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Cell Biology

Article Title: Formin follows function: a muscle-specific isoform of FHOD3 is regulated by CK2 phosphorylation and promotes myofibril maintenance

doi: 10.1083/jcb.201005060

Figure Lengend Snippet: The muscle-specific FHOD3 isoform is targeted to the sarcomere in a phosphorylation-dependent fashion. (A and B) NRCs were transfected with FHOD3-HA including or lacking the alternative T(D/E) 5 XE exon (A) or after mutation of residues T1474 and T1476 to alanine (T>A) or aspartate (T>D; B). After 48 h, cells were fixed and stained with monoclonal rat anti-HA and pRb anti–myosin-binding protein C (MyBP-C). Constructs including the T(D/E) 5 XE exon targeted to the A band, whereas constructs lacking the T(D/E) 5 XE exon formed cytoplasmic aggregates. Mutation of the threonine residues to alanine induced the formation of cytoplasmic aggregates as well. No change in localization was seen after mutation of the threonine residues to aspartic acid. (C) Untransfected NRCs were treated with 10 µM of the CK2 inhibitor DMAT for 24 h and then fixed and stained with anti-FHOD3 and anti–α-actinin antibodies. Untreated cells were used as a control. Similar to exogenous FHOD3, endogenous FHOD3 localizes to the A band but forms aggregates when phosphorylation by CK2 is inhibited.

Article Snippet: For in cyto experiments, COS-1 cells or NRCs were grown for 24 h and then treated with 10 μM of the CK2 Inhibitor DMAT (InSolution; Merck Chemicals) for 24 h before cell lysis or fixation and antibody staining as described earlier.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Staining, Binding Assay, Construct, Control

Journal: The Journal of Cell Biology

Article Title: Formin follows function: a muscle-specific isoform of FHOD3 is regulated by CK2 phosphorylation and promotes myofibril maintenance

doi: 10.1083/jcb.201005060

Figure Lengend Snippet: FHOD3 interacts with the p62-PB1 domain in a phosphorylation-regulated manner. (A) Yeast two-hybrid assay to test for the specificity and strength of the p62 interaction with the FHOD3-FH2 domain. p62-pGAD10 was transformed into yeast together with FH2L and FH2S-LexA, as well as with the empty LexA vector as a control, and grown on medium containing 0, 0.5, and 5.0 mM of 3-AT. p62 strongly interacts with FH2S and interacts much more weakly with FH2L. (B) GST pull-down assays to analyze the FHOD3–p62 interaction. Equal amounts of purified FH2L-GST, FH2S-GST, or CK2-phosphorylated FH2L-GST (CK2-P) were incubated with lysates of p62-GFP–transfected COS-1 cells and subjected to immunoblotting with monoclonal mouse (mM) anti-GFP and, subsequently, polyclonal goat anti-GST. The successful phosphorylation of FH2L-GST was controlled by incubation with monoclonal mouse anti–phospho-threonine (pThr). Although the presence of the unphosphorylated T(D/E) 5 XE exon has no effect on the interaction with p62, CK2 phosphorylation of FH2L-GST before the incubation strongly reduces the interaction. Inp, input. (C) Coimmunoprecipitation (IP; CoIP) of full-length FHOD3 and p62. Lysates of COS-1 cells, which were transfected with p62-GFP and FHOD3-HA constructs containing (FHOD3L) or excluding (FHOD3S) the T(D/E) 5 XE exon, as well as with constructs with residues T1474 and T1476 mutated to alanine (FHOD3A) or aspartic acid (FHOD3D), were immunoprecipitated with anti-HA and subjected to immunoblotting with anti-GFP and subsequently anti-HA antibodies. Mutation of the threonine residues to aspartic acid, but not to alanine, greatly reduced the interaction. (D) p62 domain structure and transfection constructs. Domains were constructed according to Simple Modular Architecture Research Tool (SMART) and . ZZ, zinc finger domain (ZZ type). (E) To identify the minimal binding site, COS-1 cells were cotransfected with FHOD3S-HA and p62-GFP constructs containing the PB1 domain, the UBA domain, or the protein without the UBA domain (ΔUBA) as indicated in D. Lysates were treated as described earlier. Binding was found with the ΔUBA and the PB1 domain, indicating that binding of FHOD3 requires presence of the PB1 domain in p62. (F) Cotransfection of NRCs with p62-HA and FHOD3-GFP constructs. Cells were stained with monoclonal rat anti-HA. p62 recruits FHOD3 lacking the T(D/E) 5 XE exon into aggregates. (G) Transfection of NRCs with FHOD3-GFP constructs and staining with pRb anti-p62 show colocalization of endogenous p62 only with FHOD3 lacking the T(D/E) 5 XE exon. (H) Visualization of endogenous FHOD3 and p62 by immunofluorescence in NRCs that were treated with DMAT to inhibit CK2 demonstrates colocalization after the redistribution of FHOD3.

Article Snippet: For in cyto experiments, COS-1 cells or NRCs were grown for 24 h and then treated with 10 μM of the CK2 Inhibitor DMAT (InSolution; Merck Chemicals) for 24 h before cell lysis or fixation and antibody staining as described earlier.

Techniques: Phospho-proteomics, Y2H Assay, Transformation Assay, Plasmid Preparation, Control, Purification, Incubation, Transfection, Western Blot, Construct, Immunoprecipitation, Mutagenesis, Binding Assay, Cotransfection, Staining, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: Formin follows function: a muscle-specific isoform of FHOD3 is regulated by CK2 phosphorylation and promotes myofibril maintenance

doi: 10.1083/jcb.201005060

Figure Lengend Snippet: Model for the regulation and subcellular localization of different FHOD3 isoforms. The insertion of the T(D/E) 5 XE exon into muscle FHOD3 is indicated by the yellow triangle at the end of the FH2 domain. Phosphorylation by CK2 abolishes the interaction of muscle FHOD3 with p62 and promotes targeting to the myofibrils.

Article Snippet: For in cyto experiments, COS-1 cells or NRCs were grown for 24 h and then treated with 10 μM of the CK2 Inhibitor DMAT (InSolution; Merck Chemicals) for 24 h before cell lysis or fixation and antibody staining as described earlier.

Techniques: Phospho-proteomics