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SUMOylation of nuclear actin.

Hofmann WA, Arduini A, Nicol SM, Camacho CJ, Lessard JL, Fuller-Pace FV, de Lanerolle P - J. Cell Biol. (2009)

Bottom Line: Nuclear actin is involved in a variety of nuclear processes including transcription, chromatin remodeling, and intranuclear transport.We now show that nuclear actin is modified by SUMO2 and SUMO3 and that computational modeling and site-directed mutagenesis identified K68 and K284 as critical sites for SUMOylating actin.We also present a model for the actin-SUMO complex and show that SUMOylation is required for the nuclear localization of actin.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL 60612, USA.

ABSTRACT
Actin, a major component of the cytoplasm, is also abundant in the nucleus. Nuclear actin is involved in a variety of nuclear processes including transcription, chromatin remodeling, and intranuclear transport. Nevertheless, the regulation of nuclear actin by posttranslational modifications has not been investigated. We now show that nuclear actin is modified by SUMO2 and SUMO3 and that computational modeling and site-directed mutagenesis identified K68 and K284 as critical sites for SUMOylating actin. We also present a model for the actin-SUMO complex and show that SUMOylation is required for the nuclear localization of actin.

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Actin is SUMOylated in vivo. (A and B) COS-7 cells were cotransfected with His-tagged SUMO proteins and various myc-tagged actin constructs. His-tagged proteins were purified on a Ni2+ column and analyzed with a myc antibody. Myc-actin (A) and myc-NLS-actin (B) are modified in vivo predominantly by SUMO 2 and 3. Modification of actin by SUMO2 and SUMO3 is increased when the amount of nuclear actin is increased (compare A and B). Vector indicates cells that were only transfected with myc-actin or myc-NLS-actin. (C) SUMOylation of nuclear targeted actin: COS-7 cells were cotransfected with His-SUMO2 and either myc-actin, myc-NLS-actin, or myc-actin-NES-1. Myc-NLS-actin and myc-actin-NES-1 are targeted to the nucleus via the NLS or are retained in the nucleus because of a mutation in the NES. The two actin constructs that are targeted to the nucleus show strong SUMOylation. In contrast, myc-actin, which is predominantly cytoplasmic, shows weaker modification. Molecular mass markers are indicated in kD. Asterisks, SUMOylated actin.
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fig2: Actin is SUMOylated in vivo. (A and B) COS-7 cells were cotransfected with His-tagged SUMO proteins and various myc-tagged actin constructs. His-tagged proteins were purified on a Ni2+ column and analyzed with a myc antibody. Myc-actin (A) and myc-NLS-actin (B) are modified in vivo predominantly by SUMO 2 and 3. Modification of actin by SUMO2 and SUMO3 is increased when the amount of nuclear actin is increased (compare A and B). Vector indicates cells that were only transfected with myc-actin or myc-NLS-actin. (C) SUMOylation of nuclear targeted actin: COS-7 cells were cotransfected with His-SUMO2 and either myc-actin, myc-NLS-actin, or myc-actin-NES-1. Myc-NLS-actin and myc-actin-NES-1 are targeted to the nucleus via the NLS or are retained in the nucleus because of a mutation in the NES. The two actin constructs that are targeted to the nucleus show strong SUMOylation. In contrast, myc-actin, which is predominantly cytoplasmic, shows weaker modification. Molecular mass markers are indicated in kD. Asterisks, SUMOylated actin.

Mentions: To further characterize the in vivo SUMOylation of actin, we cotransfected COS-7 cells with myc-actin or myc-NLS-actin and His-tagged SUMO1, SUMO2, or SUMO3 constructs. The cells were lysed 42 h after transfection, and the His-tagged SUMO proteins, which are expressed at equivalent levels under similar conditions (Jacobs et al., 2007), were pulled down using Ni-agarose beads, resolved by SDS-PAGE, transferred to nitrocellulose, and probed with anti-myc antibodies to detect if the myc-tagged actin is SUMOylated. Fig. 2 (A and B) shows that both the myc-actin and the myc-NLS-actin are modified in cells by SUMO2 and SUMO3 but not to a significant level by SUMO1. This corroborates the results in Fig. 1 A that demonstrate that actin is preferably modified by SUMO 2 and 3 in vitro. Similarly, Ni pull-down experiments on cells expressing myc-actin (Fig. 2 A) and myc-NLS-actin (Fig. 2 B) corroborated the results in Fig. 1 B by demonstrating that actin that is targeted to the nucleus (myc-NLS-actin) is SUMOylated and poly-SUMOylated to a greater extent than the myc-actin, probably because most of the myc-actin is in the cytoplasm.


SUMOylation of nuclear actin.

Hofmann WA, Arduini A, Nicol SM, Camacho CJ, Lessard JL, Fuller-Pace FV, de Lanerolle P - J. Cell Biol. (2009)

Actin is SUMOylated in vivo. (A and B) COS-7 cells were cotransfected with His-tagged SUMO proteins and various myc-tagged actin constructs. His-tagged proteins were purified on a Ni2+ column and analyzed with a myc antibody. Myc-actin (A) and myc-NLS-actin (B) are modified in vivo predominantly by SUMO 2 and 3. Modification of actin by SUMO2 and SUMO3 is increased when the amount of nuclear actin is increased (compare A and B). Vector indicates cells that were only transfected with myc-actin or myc-NLS-actin. (C) SUMOylation of nuclear targeted actin: COS-7 cells were cotransfected with His-SUMO2 and either myc-actin, myc-NLS-actin, or myc-actin-NES-1. Myc-NLS-actin and myc-actin-NES-1 are targeted to the nucleus via the NLS or are retained in the nucleus because of a mutation in the NES. The two actin constructs that are targeted to the nucleus show strong SUMOylation. In contrast, myc-actin, which is predominantly cytoplasmic, shows weaker modification. Molecular mass markers are indicated in kD. Asterisks, SUMOylated actin.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC2717643&req=5

fig2: Actin is SUMOylated in vivo. (A and B) COS-7 cells were cotransfected with His-tagged SUMO proteins and various myc-tagged actin constructs. His-tagged proteins were purified on a Ni2+ column and analyzed with a myc antibody. Myc-actin (A) and myc-NLS-actin (B) are modified in vivo predominantly by SUMO 2 and 3. Modification of actin by SUMO2 and SUMO3 is increased when the amount of nuclear actin is increased (compare A and B). Vector indicates cells that were only transfected with myc-actin or myc-NLS-actin. (C) SUMOylation of nuclear targeted actin: COS-7 cells were cotransfected with His-SUMO2 and either myc-actin, myc-NLS-actin, or myc-actin-NES-1. Myc-NLS-actin and myc-actin-NES-1 are targeted to the nucleus via the NLS or are retained in the nucleus because of a mutation in the NES. The two actin constructs that are targeted to the nucleus show strong SUMOylation. In contrast, myc-actin, which is predominantly cytoplasmic, shows weaker modification. Molecular mass markers are indicated in kD. Asterisks, SUMOylated actin.
Mentions: To further characterize the in vivo SUMOylation of actin, we cotransfected COS-7 cells with myc-actin or myc-NLS-actin and His-tagged SUMO1, SUMO2, or SUMO3 constructs. The cells were lysed 42 h after transfection, and the His-tagged SUMO proteins, which are expressed at equivalent levels under similar conditions (Jacobs et al., 2007), were pulled down using Ni-agarose beads, resolved by SDS-PAGE, transferred to nitrocellulose, and probed with anti-myc antibodies to detect if the myc-tagged actin is SUMOylated. Fig. 2 (A and B) shows that both the myc-actin and the myc-NLS-actin are modified in cells by SUMO2 and SUMO3 but not to a significant level by SUMO1. This corroborates the results in Fig. 1 A that demonstrate that actin is preferably modified by SUMO 2 and 3 in vitro. Similarly, Ni pull-down experiments on cells expressing myc-actin (Fig. 2 A) and myc-NLS-actin (Fig. 2 B) corroborated the results in Fig. 1 B by demonstrating that actin that is targeted to the nucleus (myc-NLS-actin) is SUMOylated and poly-SUMOylated to a greater extent than the myc-actin, probably because most of the myc-actin is in the cytoplasm.

Bottom Line: Nuclear actin is involved in a variety of nuclear processes including transcription, chromatin remodeling, and intranuclear transport.We now show that nuclear actin is modified by SUMO2 and SUMO3 and that computational modeling and site-directed mutagenesis identified K68 and K284 as critical sites for SUMOylating actin.We also present a model for the actin-SUMO complex and show that SUMOylation is required for the nuclear localization of actin.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL 60612, USA.

ABSTRACT
Actin, a major component of the cytoplasm, is also abundant in the nucleus. Nuclear actin is involved in a variety of nuclear processes including transcription, chromatin remodeling, and intranuclear transport. Nevertheless, the regulation of nuclear actin by posttranslational modifications has not been investigated. We now show that nuclear actin is modified by SUMO2 and SUMO3 and that computational modeling and site-directed mutagenesis identified K68 and K284 as critical sites for SUMOylating actin. We also present a model for the actin-SUMO complex and show that SUMOylation is required for the nuclear localization of actin.

Show MeSH