Identification and characterization of a protein containing formin homology (FH1/FH2) domains

Westendorf, Jennifer J.; Mernaugh, Ray; Hiebert, Scott W.

doi:10.1016/s0378-1119(99)00127-4

Cited by 51 publications

(66 citation statements)

References 28 publications

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“…Plasmids-The pCMV5-HA-FHOS full-length and HA-FHOS-(1-1010) expression plasmids were previously described (30 Prokaryotic expression vectors for the GST-GTPase fusion proteins were kindly provided by Shuh Narumiya, pGEX-2T-RhoA, -Rac1, and Cdc42 (2); Gary Bokoch, pGEX-4T-Rac2 (31); Nora Heisterkamp, pGEX-2T-Rac3 (32); and Ulla Knaus, pGEX-Rac132 and -Rac 231 (33). pZipNeo-RacN17, Cdc42N17, and RhoAN19 expression vectors were obtained from Yi Zheng (34).…”

Section: Methodsmentioning

confidence: 99%

“…Immune complexes were collected with 50 l of 50% protein G-Sepharose slurry for 2 h at 4°C, washed three times with modified RIPA buffer, resolved by 12% SDS-PAGE, and transferred to Immobilon P (Millipore). Rac1 and FHOS were detected by immunoblotting with murine Rac1 (1:1000, UBI) and FHOS-(1:600) (30) antibodies diluted in Tris-buffered saline containing 0.1% Tween 20 and 5% non-fat dried milk. Proteins were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and autoradiography.…”

Section: Fhos-gtpase Interaction Assays-gst-gtpase Fusion Proteinsmentioning

confidence: 99%

“…For yeast two-hybrid assays S. cerevisiae strain Y190 (MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3, Ϫ112 ϩURA3::GAL3lacZ, LYS2::GAL3 HIS3) were co-transformed with the indicated pAS-2-1-FHOS expression plasmid and pACT-FHOS-(904 -1165) (30). Transformed yeast were selected on SD media lacking leucine (Leu), tryptophan (Trp), and histidine (His).…”

Section: Fhos-gtpase Interaction Assays-gst-gtpase Fusion Proteinsmentioning

confidence: 99%

“…Cells were mounted in 90% glycerol containing 0.4% n-propyl gallate. Endogenous FHOS was detected in HeLa cells with FHOS antiserum (30). DNA was counterstained for 5 min with Hoescht's dye (5 g/ml) as indicated.…”

Section: Fhos-gtpase Interaction Assays-gst-gtpase Fusion Proteinsmentioning

confidence: 99%

“…FHOS is a member of the formin homology (FH) family of proteins that is expressed at high levels in human splenic cells (30). FHOS contains all the conserved domains, including FH1, FH2, and FH3 domains, and a coiled-coil, that are characteristic of FH proteins.…”

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confidence: 99%

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The Formin/Diaphanous-related Protein, FHOS, Interacts with Rac1 and Activates Transcription from the Serum Response Element

Westendorf

2001

Journal of Biological Chemistry

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107

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FHOS is a member of the formin homology (FH) family of proteins and is expressed at high levels in splenic cells. FH proteins link cellular signaling pathways to the actin cytoskeleton and serum response factor-dependent transcription. In these studies, the role of FHOS in Rho family GTPase signaling pathways was analyzed. FHOS interacted with the polybasic domain in the Rac1 C terminus in a guanine nucleotideindependent manner but did not interact with RhoA, Cdc42Hs, Rac2, or Rac3. Intramolecular autoinhibitory interactions between the C terminus of FHOS and an N-terminal region partially overlapping the Rac1 interaction domain were also identified. FHOS truncation mutants lacking the N-or C-terminal autoregulatory domains stimulated transcription of a c-fos serum response element (SRE)-driven reporter. Overexpression of wild-type and mutant (N17 and V12) Rac1 proteins repressed SRE induction by the N-terminal FHOS deletion mutant but not by the C-terminal FHOS deletion mutant. Immunofluorescence studies indicated that the localization of the mutant FHOS proteins might contribute to their differential responses to Rac1. Wild-type FHOS and the N-terminal deletion mutant localized to the perinuclear region and membrane edges. In contrast, the C-terminal FHOS mutants were diffusely localized. These data suggest that FHOS induces transcription from SREs by multiple pathways and that Rac1 may influence the course of some FHOSinduced signaling events.

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Section: Methodsmentioning

confidence: 99%

Section: Fhos-gtpase Interaction Assays-gst-gtpase Fusion Proteinsmentioning

confidence: 99%

Section: Fhos-gtpase Interaction Assays-gst-gtpase Fusion Proteinsmentioning

confidence: 99%

Section: Fhos-gtpase Interaction Assays-gst-gtpase Fusion Proteinsmentioning

confidence: 99%

mentioning

confidence: 99%

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The Formin/Diaphanous-related Protein, FHOS, Interacts with Rac1 and Activates Transcription from the Serum Response Element

Westendorf

2001

Journal of Biological Chemistry

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107

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The Formin FHOD1 in Cardiomyocytes

Dwyer

Pluess

Iskratsch

et al. 2014

The Anatomical Record

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Members of the formin family are known to be involved in the regulation of the actin cytoskeleton. We have recently identified a muscle specific splice variant of the formin FHOD3 and demonstrated its role in the maintenance of the contractile filaments of cardiomyocytes. Here, we characterize the expression and subcellular localization of FHOD3's closest relative, FHOD1, in the heart. Confocal microscopy shows that FHOD1 is mainly located at the intercalated disc, the special type of cell-cell contact between cardiomyocytes, but also partially associated with the myofibrils. Subcellular targeting of FHOD1 is probably mediated by its N-terminal domain, since expression constructs lacking this domain show aberrant localization in primary cultures of neonatal rat cardiomyocytes. Finally, we show that in contrast to FHOD3, FHOD1 shows increased expression levels in dilated cardiomyopathy, suggesting that the two formins play distinct roles and are differentially regulated in cardiomyocytes. Anat Rec, 297:1560Rec, 297: -1570Rec, 297: , 2014. V C 2014 Wiley Periodicals, Inc.Key words: formin; actin; intercalated disc; sarcomere Cardiomyocytes, which make up the bulk of heart mass, are characterized by an exquisitely organized cytoskeleton, which is the basis of maximal functional output (Ehler, 2010). The actin cytoskeleton comprises the thin filaments in the myofibrils, which mediate muscle contraction together with the thick (myosin) filaments, but in addition plays also a role in the organization of the plasma membrane cytoskeleton as well as in the anchoring of myofibrils at the specialized type of cell-cell contact in heart tissue, the intercalated disc (Dwyer et al., 2012). Despite this crucial role of actin in the function of cardiomyocytes, it is subjected to an impressive amount of protein turnover. Measurements in rat hearts have suggested that actin has a half-life of about 10 days (Martin, 1981). How the exchange of actin molecules is achieved in the continuously working cardiomyocyte is not well understood.We have recently focused our attention on a potential role of formins in the heart. Formins are proteins, which are mainly involved in the formation of linear actin filaments (Chesarone et al., 2010). They are characterized by the possession of an FH1 and an FH2 domain (for domain layout of formins see Fig. 1), which together make up the actin capturing and polymerization machinery and so far 15 different formins were described in mammals (Sch€ onichen and Geyer, 2010).One characteristic of formins is that they normally exist in an autoinhibited state, which is brought about Abbreviations used: DAD 5 Diaphanous autoregulatory domain; DCM 5 dilated cardiomyopathy; DID 5 Diaphanous inhibitory domain; FH 5 formin homology domain; GBD 5 GTPase binding domain; NRC 5 neonatal rat cardiomyocytes.

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The multiplicity of human formins: Expression patterns in cells and tissues

et al. 2013

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Formins are actin binding proteins conserved across species from plants to humans. The formin family is defined by their common FH2 domains. The 15 distinct human formins are involved in a broad range of cellular functions, including cell adhesion, cytokinesis, cell polarity, and cell morphogenesis. Their commonality is actin polymerization activity inherent to FH2 domains. While still requiring much study, biochemical activity of formins has been carefully described. In contrast, much less is known of their activities in complex living systems. With the diversity of the formin family and the actin structures that they affect, an extensive future of study beckons. In this study, we report the expression level of all 15 formins in 22 different human cell and tissue types using quantitative real-time PCR (qPCR). Identification of major themes in formin expression and documentation of expression profiles should facilitate the cellular study of formins.

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Identification and characterization of a protein containing formin homology (FH1/FH2) domains

Cited by 51 publications

References 28 publications

The Formin/Diaphanous-related Protein, FHOS, Interacts with Rac1 and Activates Transcription from the Serum Response Element

The Formin/Diaphanous-related Protein, FHOS, Interacts with Rac1 and Activates Transcription from the Serum Response Element

The Formin FHOD1 in Cardiomyocytes

The multiplicity of human formins: Expression patterns in cells and tissues

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