Mitotic-dependent phosphorylation of leukemia-associated RhoGEF (LARG) by Cdk1

Abstract

Rho GTPases are integral to the regulation of actin cytoskeleton-dependent processes, including mitosis. Rho and leukemia-associated Rho guanine-nucleotide exchange factor (LARG), also known as ARHGEF12, are involved in mitosis as well as diseases such as cancer and heart disease. Since LARG has a role in mitosis and diverse signaling functions beyond mitosis, it is important to understand the regulation of the protein through modifications such as phosphorylation. Here we report that LARG undergoes a mitotic-dependent and cyclin-dependent kinase 1 (Cdk1) inhibitor-sensitive phosphorylation. Additionally, LARG is phosphorylated at the onset of mitosis and dephosphorylated as cells exit mitosis, concomitant with Cdk1 activity. Furthermore, using an in vitro kinase assay, we show that LARG can be directly phosphorylated by Cdk1. Through expression of phosphonull mutants that contain non-phosphorylatable alanine mutations at potential Cdk1 S/TP sites, we demonstrate that LARG phosphorylation occurs in both termini. Using phosphospecific antibodies, we confirm that two sites, serine 190 and serine 1176, are phosphorylated during mitosis in a Cdk1-dependent manner. In addition, these phosphospecific antibodies show phosphorylated LARG at specific mitotic locations, namely the mitotic organizing centers and flanking the midbody. Lastly, RhoA activity assays reveal that phosphonull LARG is more active in cells than phosphomimetic LARG. Our data thus identifies LARG as a phosphoregulated RhoGEF during mitosis.

Keywords: Cdk1; Cytokinesis; Mitosis; Phosphorylation; RhoA; RhoGEF; Subcellular localization.

Figure 1. Mitotic-dependent phosphorylation of LARG

(A) Mitotic-dependent mobility shift of LARG. HeLa cells were treated with nocodazole as described in Materials and Methods. Cells arrested in prometaphase (M) were collected by mitotic shake-off, and cells remaining on plates were collected and designated as G2 cells. Lysates from asynchronous (A), G2, or prometaphase-arrested (M) HeLa cells were immunoblotted with anti-LARG antibody. (B) Phosphatase treatment reverses mitotic-dependent phosphorylation of LARG. G2 or prometaphase-arrested (M) lysates from HeLa (left panels) and COS-7 (right panels) cells were prepared as above in A. Lysates from prometaphase-arrested (M) cells were then treated with or without λ protein phosphatase, followed by immunoblotting with anti-LARG antibody or anti-GAPDH antibody (loading control).

Figure 2. LARG is phosphorylated and dephosphorylated during mitosis

(A) LARG is dephosphorylated as cells exit mitosis. HeLa cells arrested in prometaphase by nocodazole were collected by mitotic shake-off and re-plated on poly-L-lysine coated plates in fresh media without nocodazole to release the cells from mitotic arrest. Cells were lysed at the indicated times after release, and lysates were immunoblotted using anti-LARG, anti-cyclin B1 and anti-HSP90 (loading control) antibodies. (B) LARG is rapidly phosphorylated after release from Cdk1 inhibitor RO-3306. Cells were synchronized with RO-3306, released from G2/M block by washing and incubating in media without RO-3306, and lysed at indicated times. Cell lysates were immunoblotted using anti-LARG and anti-HSP90 (loading control) antibodies. (C) LARG is phosphorylated coordinate with Cyclin B1 accumulation and is dephosphorylated in late mitosis. HeLa cells were treated with 2 mM thymidine for 16 hrs to block the cells in S phase, followed by release in normal growth medium for 8 hrs and subsequent thymidine treatment for 16 hrs. HeLa cells were then released from S-phase block into normal growth media and collected at indicated time points. Cell lysates were immunoblotted using anti-LARG, anti-cyclin B1, anti-GAPDH (loading control) antibodies.

Figure 3. LARG is phosphorylated by Cdk1 during mitosis

(A) Inhibition of Cdk1 abolishes the slower mobility of mitotic LARG. Mitotic cells, arrested after nocodazole treatment, were collected and re-plated on poly-L-lysine in the continued presence of nocodazole and in presence of the indicated mitotic kinase inhibitors or Calyculin A or vehicle control (DMSO) for 30 or 60 minutes. Cell lysates were immunoblotted using anti-LARG antibody. (B) Full-length LARG and ΔN LARG are phosphorylated in vitro by Cdk1. Purified proteins (1 μM) from Sf9-baculovirus system (right panel) were incubated with CDK1-cyclin B (13 nM) in incubation buffer (containing 20 μM [gamma-32P]ATP) for 5 min at 30°C. Samples were analyzed by SDS-PAGE followed by autoradiography (left panel). (C) Domain structure of LARG with potential Cdk1 Sites. Amino acid numbering is indicated in the above figure. Domains: PDZ, post synaptic density 95/disc-large/zona occludens; RGS, regulator of G protein signalling; DH, Dbl homology; PH, pleckstin homology; NLS, nuclear localization signal; CC, coiled-coil. The pink stars indicate potential Cdk1 sites. The dark blue stars indicate the sites that are the epitopes targeted by the pS190 and pS1176 phosphospecific antibodies. (D) Mitotic-dependent gel mobility shift of LARG is altered by N- and C-terminal phosphonull mutations. HEK293 cells were transfected with the indicated constructs, treated with nocodazole or vehicle control. Nocodazole-treated cells were subjected to mitotic shake-off, and cell lysates were prepared (M). Cell lysates were also prepared from asynchronously growing cells (A). Lysates were then immunoblotted using an anti-GFP antibody.

Figure 4. Phosphospecific antibodies reveal that LARG is phosphorylated at S190 and S1176 during mitosis

(A) Phosphoantibody specificity for phosphorylated LARG was determined by phosphatase treatment of mitotic cell lysates. Cell lysates were prepared from asynchronous HeLa cells (A) and nocodazole-arrested mitotic HeLa cells (M). As described in Materials and Methods, mitotic cell lysates were treated with λ protein phosphatase (+). Then lysates were immunoblotted with anti-LARG, anti-pS190 LARG, and anti-pS1176 antibodies, as indicated. (B) Phosphoantibody specificity for LARG was determined by siRNA knockdown of LARG. HeLa cells were treated with non-specific (ns) siRNA and siRNA specific for LARG (siLARG), as described previously [9]. After nocodazole treatment to arrest cells in prometaphase, mitotic cells were harvested by shake-off and lysates were prepared (M). Lysates were also prepared from the G₂/interphase cells remaining on the culture dish (I). Lysates were then immunoblotted with anti-LARG, anti-pS190 LARG, and anti-pS1176 LARG antibodies, as indicated. (C) Immunoblot showing efficiency of LARG siRNA. Lysates were prepared from G₂/interphase (G2) and mitotic cells (M) treated with Ns-siRNA or LARG siRNA, as described above and used in (B), followed by immunoblotting with an anti-LARG antibody. Ns lysates were used at 1, 0.5 and 0.25 equivalents compared to siLARG lysates to show efficient knockdown (≥90%) of endogenous LARG. (D and E) Phosphoantibodies were tested on LARG point and multi-site phosphomutants. HeLa cells were transfected with the indicated constructs, treated with nocodazole, and subjected to mitotic shake-off. Cell lysates from the mitotic cells (M) and asynchronous cells (A) were prepared and immunoblotted using anti-pS190 LARG, anti-pS1176 LARG and anti-HSP90 (loading control) antibodies, as indicated.

Figure 5. LARG phosphorylated at sites S190 and S1176 localizes to the centrosomes and flanks the midbody in cytokinesis

(A) HeLa cells were fixed and stained with anti-α-tubulin antibody (red), anti-pS190 LARG (green), and DNA was visualized by DAPI staining (blue). (B) Same as in left panel, except anti-pS1176 LARG is in green. Cells in different stages of mitosis were identified based on hallmark nuclear and microtubule staining.

Figure 5. LARG phosphorylated at sites S190 and S1176 localizes to the centrosomes and flanks the midbody in cytokinesis

(A) HeLa cells were fixed and stained with anti-α-tubulin antibody (red), anti-pS190 LARG (green), and DNA was visualized by DAPI staining (blue). (B) Same as in left panel, except anti-pS1176 LARG is in green. Cells in different stages of mitosis were identified based on hallmark nuclear and microtubule staining.

Figure 6. Decreased activity of phosphomimetic mutant of LARG

(A) LARG activity was assessed via serum response element (SRE) luciferase activity. HEK293 cells were transfected with expression vectors for GFP-LARG, GFP-LARG-N/CpNull, GFP-LARG-N/CpMim or control pEGFP, along with both SRE-luciferase and Renilla luciferase. SRE-luciferase activity was measured and normalized as described under Materials and Methods. The graph shows quantitation (average ± SEM, n=3) of multiple experiments. Statistical significance is represented as follows: LARG compared to N/CpNull (*, p<0.05, t-test); N/CpNull compared to N/CpMim: (**, p<0.01, t-test); and LARG compared to N/CpMim: (***, p<0.0001, t-test). (B) Pull-down of LARG using GST-G17A-RhoA. The indicated GFP-tagged LARG proteins were expressed in HEK293 cells. Cell lysates were prepared and active LARG was pulled down using GST-G17A-RhoA. LARG in the affinity pulldown (upper panel) and in the cell lysate (lower panel) was determined by immunoblotting with a GFP antibody. A representative blot is shown. The graph shows quantitation (average ± SEM, n=4) of multiple experiments, in which the amount of LARG in the GST-G17A-Rho pulldown was normalized by the total expressed LARG in the lysate. The asterisk indicates statistical significance (p<0.05, t-test) of the N/CpMim mutant compared to wt LARG and to the N/CpNull mutant.

Figure 7. Proposed model of phosphoregulation of LARG in mitosis

LARG is phosphorylated and dephosphorylated during mitosis, which regulates the GEF activity. Cdk1 phosphorylates LARG in early mitosis (G2/M) resulting in decreased GEF activity. LARG is then dephosphorylated, relieving the inhibition of GEF activity, as cells proceed past the anaphase transition towards mitotic exit. See text for further discussion.