SUMOylation regulates nucleo-cytoplasmic shuttling of Elk-1

Abstract

The transcription factor Elk-1 is a nuclear target of mitogen-activated protein kinases and regulates immediate early gene activation by extracellular signals. We show that Elk-1 is also conjugated to SUMO on either lysines 230, 249, or 254. Mutation of all three sites is necessary to fully block SUMOylation in vitro and in vivo. This Elk-1 mutant, Elk-1(3R), shuttles more rapidly to nuclei of Balb/C cells fused to transfected HeLa cells. Coexpression of SUMO-1 or -2 strongly reduces shuttling by Elk-1 without affecting that of Elk-1(3R), indicating that SUMOylation regulates nuclear retention of Elk-1. Accordingly, overexpression of Elk-1(3R) in PC12 cells, where cytoplasmic relocalization of Elk-1 has been linked to differentiation, enhances neurite extension relative to Elk-1. The effect of Elk-1, but not of the 3R mutant, was blocked upon cotransfection with SUMO-1 or -2 and enhanced by coexpression with mutant Ubc-9. Thus, SUMO conjugation is a novel regulator of Elk-1 function through the control of its nuclear-cytoplasmic shuttling.

Figure 1.

In vitro SUMOylation of Elk-1 is blocked by mutating all three consensus sites. (A) The major functional domains in human Elk-1 are indicated. Shown below is the consensus site for Ubc-9 binding and SUMOylation ψKxE, where ψ is an aliphatic amino acid and K is the lysine conjugated to SUMO, as are the sequences surrounding the three major consensus sites in Elk-1, centered at lysines 230, 249, and 254. (B) ³⁵S-labeled Elk-1, PML, or p38α were incubated with GST or GST-Ubc9 bound to glutathione agarose. After washing, bound proteins were separated by SDS-PAGE and visualized by autoradiography of the dried gel. Input indicates 10% of the ³⁵S-protein added to the incubation. White lines indicate that intervening lanes have been spliced out. (C) ³⁵S-Elk-1, either WT or mutated in each consensus site (as shown below the panel) was incubated with the indicated components. The reactions were analyzed by SDS-PAGE as in B. Elk-1 and SUMOylated Elk-1 bands are indicated. The white line indicates that intervening lanes were spliced out. (D) Elk-1 WT and the triple mutant 3R were tested for conjugation to SUMO-1 (top), -2 (middle), or -3 (bottom) as in C.

Figure 2.

Conjugation of all three SUMO isoforms to Elk-1 in transfected cells. HeLa cells were transfected with expression vectors for Elk-1 WT or 3R, together with vectors for (His)SUMO-1 (A), -2 (B), or -3 (C) as indicated below the lanes. Total proteins (Input) and His-tagged proteins were purified from denaturing cell lysates, separated on SDS-PAGE and visualized by immunoblotting for Elk-1. Left, Ni-NTA bound proteins; right, total protein. (D) HeLa cells transfected with expression vectors for (His)SUMO-2 and either WT Elk-1 or the mutants K230R, K249R, K254R, or 3R were analyzed as in A–C.

Figure 3.

Shuttling of EGFP-Elk-1 WT and 3R in HeLa/Balb/C heterokaryons. (A) HeLa cells on coverslips were transfected with expression vectors for EGFP-Elk-1 WT or 3R, as indicated. Cells were fixed 36 h later and counterstained with DAPI. (B) Heterokaryons between Balb/C mouse fibroblasts and HeLa cells transfected as in A, along with pDsRed2 to visualize the heterokaryons (not depicted). Cells were fixed 60 min later and counterstained with DAPI. The arrows indicate mouse nuclei to which EGFP-Elk-1 WT (top) and 3R (bottom) have shuttled.

Figure 4.

Elk3R enhances PC12 differentiation relative to Elk-1 WT. PC12 cells were transfected with expression vectors for (HA)Elk-1 WT, 3A or 3R, or EGFP as a control. Cells were processed 48 h later. (A) Elk-1 was visualized using an anti-HA antibody and F-actin with phalloidin-CPITC. (B) Percentage of HA-positive PC12 cells presenting neurites, defined by a length at least twice that of the cell body (Estrach et al., 2002). The numbers are representative of four experiments. The error bars indicate the SEM. (C) Immunoblot detection of endogenous Elk-1 in native and denaturing lysates of PC12 cells (top), and of SUMO-1–conjugated Elk-1 in Elk-1 but not p38^MAPK immunoprecipitates from native lysates of PC12 cells (bottom). IgG denotes the signal from the antibody used for precipitation.

Figure 5.

Coexpression of SUMO blocks and of DN-Ubc9 enhances PC12 differentiation driven by Elk-1 WT. (A) PC12 cells were transfected with expression vectors for pEGFP-SUMO-1 and (HA)Elk-1 WT or 3R. Cells were processed 48 h later. Elk-1 was visualized with an anti-HA antibody. (B and C) PC12 cells were transfected with expression vectors for pEGFP (B) or pEYFP (C), (HA)Elk-1 WT or 3R, pEGFP-SUMO-1 (B) or -2 (C) as indicated, and processed as in A. Percentage of HA- and GFP-positive PC12 cells presenting neurites, as defined in the legend to Fig. 4 B. The numbers are representative of three experiments. (D) PC12 cells were transfected with expression vectors for HA-DN-Ubc9 and pEGFP Elk-1 WT or 3R as indicated and processed as described above. The numbers are representative of three experiments. The error bars indicate the SEM.