Permanent farnesylation of lamin A mutants linked to progeria impairs its phosphorylation at serine 22 during interphase

Intranuclear distribution of progerin depends on serine 22 phosphorylation

To study the biology and turnover of progerin, the lamin A mutant protein responsible for Hutchinson-Gilford progeria syndrome, we used retroviral vectors that express the wild type prelamin A, mature lamin A (mimicking the protein obtained after cleavage by ZMPSTE24) and mutant derivatives (Figure 1A). Expression of progerin in U-2 OS cells leads to an accumulation of this protein in the nuclear envelope and deformation of the nucleus (Figure 1B). Mutating the cysteine residue in the farnesylation site of progerin to serine (Progerin CS) abolished its ability to induce nuclear deformation and led to three distinct patterns of intranuclear distribution of progerin (Figure 1C). Most cells have a homogenous staining in the nucleoplasm and the nuclear periphery (Figure 1C, center). This pattern is typical of endogenous lamin A staining reported in HeLa cells and reflects functions of lamin A both at the nuclear envelope and the nucleoplasm [5]. A large proportion of the cells display an intranuclear reticulum mainly composed of lamin rods and 7% of the cells have distinct foci (Figure 1C, top and bottom respectively). Mutation of serine 22 to alanine (S22A) blocked phosphorylation at this residue and changed the intranuclear distribution of the protein. Most cells showed either the rods pattern or homogenous staining but foci were entirely absent (Figure 1C). Mutation of serine 22 to aspartic acid (S22D) to mimic phosphorylation had a dramatic consequence for Progerin CS distribution and 100% of the cells displayed round foci (Figure 1C). These patterns suggest that Progerin CS foci are associated to serine 22 phosphorylation.

We were not able to detect endogenous lamin A in the nuclear lamina in cells with foci of Progerin CS probably because non-farnesylated progerin transits through the nuclear lamina, interacts with lamin A and carries it to the foci. To confirm the idea that Progerin CS can interact with wild type lamin A and recruit it to foci, we cotransfected U-2 OS cells with GFP-pre-lamin A and RFP-progerin CS and verified their localization using fluorescence microscopy. We found that non-farnesylated progerin and lamin A-GFP colocalized in foci (Figure 1D).

We next studied the phosphorylation of progerin and progerin CS at serines 22 and 392 in U-2 OS cells using phospho-specific antibodies. In non-synchronized cells, little progerin is phosphorylated at serine 22 but phosphorylation at serine 392 was readily detectable (Figure 2A and B). Progerin CS was phosphorylated at serine 22, displaying two staining patterns of localization (homogenous staining in most of the cells and foci) (Figure 2B). Although progerin was poorly phosphorylated at serine 22, treatment with the farnesyl transferase inhibitor FTI-277 increased its S22 phosphorylation (Figure 2C and D). Taken together the results show that farnesylation prevents efficient phos-phorylation of lamin A at serine 22 in interphase cells and suggest that one role for cleavage and defarnesylation of lamin A is to facilitate its phosphorylation at serine 22.

Since FTI-277 can correct the farnesylation defect of progerin and improve its phosphorylation at serine 22, we next investigated its effects on cell growth. Consistent with previous report [23], FTI-277 at 3 μM did not significantly improve the proliferation defect imposed by overexpression of progerin in U-2 OS cells (Figure 2E and F). In fact, FTI-277 reduced the proliferation of control U-2 OS cells in this assay.

Depolymerization cooperates with defarnesylation to stimulate serine 22 phosphorylation of lamin A

Inhibition of lamin A farnesylation does not completely abolish its incorporation into the nuclear lamina [24,25]. In addition to farnesylation, lamin A uses head to tail interactions to form polymers and localize to the nuclear lamina [13]. It is also known that farnesylated proteins require a second signal for stable association to membranes [26]. Consistent with this, progerin in the nuclear lamina is farnesylated but also polymerized, suggesting that to control progerin levels and localization, in addition to defarnesylation the protein must be depolymerized. To verify this hypothesis, we created lamin A and progerin mutants in which the arginine at position 50 was replaced by a proline. This arginine in the conserved rod domain end of lamin A was shown to be critical for polymer formation [27]. Consistent with a previous report [27], R50P lamin A was not as well expressed as wild type lamin A and in some cells it did not form a continuous polymeric lamina as the wild type protein, but accumulated as foci in the nuclear envelope (Figure S1A). Introducing the phosphomimetic S22D mutation into lamin A led to the formation of nuclear foci in most cells both in the nucleoplasm and the nuclear envelope (Figure S1A).

Staining cells expressing these constructs with the anti-phosphoserine 22 antibody revealed a nucleoplasmic signal. Of note, none of the lamin A at the nuclear envelope was positive for serine 22 phosphorylation except the few foci at the nuclear envelope formed by R50P lamin A (Figure S1A). On immunoblots, a fraction of R50P prelamin A migrated at a higher molecular weight than mature lamin A (Figure S1B) suggesting that this mutation partially impairs maturation by ZMPSTE24. The phosphomimetic S22D lamin A migra-tes as mature lamin A (Figure S1B). Taken together, the results are consistent with the idea that defarnesylation promotes S22 phosphorylation in interphase cells.

Next we performed a similar mutational analysis in progerin. Progerin R50P localized mainly in the nuclear envelope similar to lamin A R50P. In contrast, Progerin CS with the R50P mutation formed intranuclear foci (Figure 3A) similar to those formed by S22D progerin CS (see Figure 1C) and reminiscent of liquid droplets formed by proteins with low complexity domains [28].

Of note, lamin A contains several regions of low complexity according to DisEMBL [29], a method based on neural networks that predicts these regions to several definitions of protein disorder (Figure S2). Foci at the nuclear envelope formed by R50P progerin were not associated with phosphory-lation at serine 22 (Figure 3A). However, intranuclear R50P Progerin CS foci were stained by the phosphospecific antibody against S22 phospholamin A (Figure 3A) and immunoblots show that the R50P mutation increased the fraction of progerin CS phosphorylated at serine 22 (Figure 3B). Note that Progerin CS formed intranuclear rods or foci only in around 50% of the cells (Figure 1C) indicating that the R50P mutation dramatically alters the behavior of this protein. Foci formed by R50P progerin CS are not stained by DAPI and look like nucleoli. However, R50P progerin CS does not colocolize with the nucleolar marker fibrillarin (Figure S3). Interestingly, R50P progerin CS foci were disrupted by treatment with 1% 1,6-hexanediol, an aliphatic alcohol that disrupts weak hydrophobic interactions (Figure 3C) [30]. These results are consistent with a model where progerin is resistant to serine 22 phosphorylation during interphase. Cleavage and defarnesylation facilitates serine 22 phospho-rylation, while the R50P mutation alone does not. However, combining defarnesylation with the R50P mutation dramatically enhances serine 22 phosphorylation of progerin probably because the R50P mutation disrupts the head to tail interactions that retain progerin CS and mature lamin A in the nuclear envelope in a way inaccessible to serine 22 kinases. Another interesting feature is that R50P progerin CS does not form rods in the nucleoplasm as progerin CS (Figure 1C and Figure 3A) suggesting that progerin CS can still form polymeric aggregates in the nucleoplasm and that the R50P mutation prevents this aggregation.

Next we evaluated the effects of R50P and CS mutations on the ability of progerin to inhibit cell growth in the tumor cell line U-2 OS. The CS mutation, but not the R50P mutation, prevented growth inhibition by progerin (Figure 3D). This is consistent with the data showing that the R50P mutation alone cannot rescue the S22 phosphorylation and localization defects of progerin. The growth phenotype correlated with the expression of the E2F target gene and DNA replication protein MCM6 (Figure 3E).

Interphase lamin A S22 kinases

The phosphorylation of serine 22 of lamin A is a function of CDK1 during mitosis [14]. However, in HeLa cells, anti-phosphoserine 22 lamin A antibody labels interphase cells without CDK1 activity [16]. To investigate which kinases may affect intranuclear lamin A during interphase, we took advantage of the foci formed by the R50P progerin CS mutant that are easily visualized with anti-lamin A/C antibodies. Serine 22 is part of a SPTR motif recognized by proline directed kinases of the CDK/MAP kinase families. In agreement, we found multiple CDK/MAP kinases colocalized with R50P progerin CS in nuclear foci. They include CDK2, CDK4, CDK6 and p38 MAPK but not MEK or ERK (Figure 4A). Unexpectedly, MSK1 and RSK also localized to these foci, suggesting that R50P progerin could be phosphorylated at other sites by these kinases as well. In cells expressing R50P progerin CS, the pan CDK inhibitor flavopiridol [31] suppressed serine 22 phosphorylation (Figure 4B). Flavopiridol did not change the total levels of progerin CS as measured with an anti-lamin A antibody. The fraction of progerin CS that is phosphorylated should correspond to the fraction that forms foci in the nucleoplasm (around 7% of the cells in Figure 1C). Hence, if inhibition of phosphorylation stabilized progerin, it will be difficult to observe this effect in progerin CS expressing cells where still a large fraction of the protein is associated to the nuclear lamina or form intranuclear aggregates.

To investigate whether inhibition of phosphorylation affects lamin A levels, we expressed mature lamin A (normally lamin A is first expressed as prelamin A which is then processed by ZMPSTE24 into the mature protein). Using this construct we can now visualize that both flavopiridol and the CDK4/6 inhibitor PD0332991 increased mature lamin A levels (Figure 4C) suggesting that CDK-dependent phosphorylation of lamin A controls its levels. As expected, both drugs inhibited lamin A phosphorylation at serine 22 as measured by normalization phospho-serine 22 lamin A levels on total lamin A levels (Figure 4C). To further investigate the role of endogenous CDK activity on endogenous lamin A levels in interphase, we used two validated shRNA against CDK6. Reducing CDK6 levels increased lamin A levels as assessed by immunoblots (Figure 4D) or immunofluorescence (Figure 4E). These results suggest that mature lamin A levels are controlled by CDK-dependent serine 22 phosphorylation in interphase.

Farnesyl transferase inhibitors increase serine 22 phosphorylation of endogenous progerin

Next we studied whether preventing farnesylation could increase the phosphorylation of endogenous progerin in cells obtained from patients with HGPS. Treatment with FTI-277 decreased the levels of endogenous progerin so we normalized the signal of serine 22 phosphorylation on the levels of progerin in cells. After this normalization, it is clear that FTI-277 increased serine 22 phosphorylation of endogenous progerin (Figures 5A and B). We conclude that absence of farnesylation facilitates the phosphorylation of endogenous lamin A at serine 22 during interphase.

To determine whether inhibition of farnesylation affected the localization of endogenous progerin, we used immunofluorescence in normal and HGPS fibroblasts treated with FTI-277. Both wild type lamin A and progerin localized to intranuclear foci after treatment with FTI-277 (Figure 5C) and these foci were stained by the anti-phospho serine 22 lamin A antibody (Figure 5D). This phosphorylation was important to control the overall levels of progerin, since treatment of HGPS cells with FTI-277 decreased progerin levels and induced intranuclear foci but both flavopiridol and PD0332991 inhibited the formation of foci and increased the levels of progerin (Figure 5E).

It is known that older cells express higher levels of CDK inhibitors such as p16INK4a [32] and p21 [33]. It is thus possible that abnormalities in HGPS increase with age or accumulate in tissues with high turnover because concomitant expression of CDK inhibitors (CKI) in these situations further impairs serine 22 phosphorylation of progerin. In agreement with this model, we recently reported that mutating serine 22 to alanine in progerin increases the ability of the protein to induce cellular senescence [34]. We thus treated HGPS cells with the CDK inhibitor flavopiridol and evaluated the number of population doublings required to attain senescence. We found that the CDK inhibitor accelerated the senescence process in HGPS fibroblasts (Figure 6A). Consistent with the senescence phenotype, cells treated with flavopiridol have lower expression of the E2F target gene MCM6 and high expression of p53. This result suggests a pathological synergism between expression of progerin and senescence-associated expression of CDK inhibitors.

Cells and compounds

Human osteosarcoma U-2 OS cells (HTB-96, ATCC), BJ fibroblasts (a gift from S. W Lowe) and fibroblasts from progeria patient (HGADFN003, Progeria Foundation) were cultured in Dulbecco's modified Eagle's medium (Wisent, St-Bruno, QC) supplemented with 10% (for U-2 OS and BJ) or 15% (for HGADFN003) fetal bovine serum (FBS) (Wisent). Flavopiridol was from (Selleckchem, http://www.selleckchem.com/). PD0332991 and FTI-277 were purchased from Sigma-Aldrich.

Retroviral vectors and retroviral-mediated gene transfer

Retroviral vector pLPC is from S. W. Lowe. pLPC wtlaminA, pLPC progerin, pLPC progerinCS, pLPC S22AprogerinCS were described in [34]. S22D point mutation was introduced into progerinCS and wild type prelamin A by site-directed mutagenesis using forward primer 5′-AGCTCCACTCCGCTGGACCCCACCCG CATCACC-3′ and reverse primer 5′-GGTGATGCG GGTGGGGTCCAGCGGAGTGGAGCT-3′. R50P point mutation (CGC→CCC) was introduced into progerin, progerinCS and wild type prelamin A expression vectors by subcloning synthetic fragments. Mature lamin A was constructed using wild type lamin A as a template and the primers: forward 5′-CTTGGATCCACCATGGAGACCCCGTCCCAGCGGCG-3′ and reverse 5′-CTTCTCGAGTTAGAGGTAGG AGCGGGTGACCAGATTGTC-3′. MLPpuroshCDK6s (Acevedo et al. Cancer Res. in press). Retroviral-mediated gene transfers were done as in [47].

Immunoblotting and immunoprecipitations

To prepare total protein lysates, cells were collected by trypsinization, washed with phosphate-buffered saline (PBS), lysed in sodium dodecyl sulfate (SDS) sample buffer (60 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 5% β-mercaptoethanol), and boiled for 5 min. For Western blotting, 25 μg of total protein were separated on SDS-polyacrylamide gel electrophoresis gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA). We used the following primary antibodies: anti-LaminA/C (2032, 1:1000, Cell Signaling), anti-phospho-Ser22LaminA (2026, 1:1000, Cell Signaling), anti-phospho-Ser392LaminA (A0503, 1:500, Asaybiotech), anti-progerin (13A4 ab66587, Abcan, 1:1000), anti-p38 (9212, 1:1000, Cell Signaling), anti-CDK6 (C21, SC-177, 1:250, Santa Cruz Biotechnology), anti-CDK2 (M-2, SC-163, 1:250, Santa Cruz Biotechnology), anti-MCM6 (provided by Dr. Heidebrecht, 1:10), anti-Tubulin (T6074, 1:5,000, Sigma). Signals were revealed after incubation with anti-mouse or anti-rabbit secondary antibodies coupled to peroxidase (Biorad) by using enhanced chemiluminescence (Amersham Pharmacia).

Fluorescence microscopy

Cells were plated on coverslips at least 24h prior to fixation with 4% paraformaldehyde in PBS for 15 minutes at room temperature. After washing with PBS, cells were permeabilized using 0.2% Triton X-100 in PBS–3% bovine serum albumin (BSA) solution for 5 min. The cells were then washed three times with PBS–3% BSA and incubated for 1-2 h at room temperature with the primary antibody: anti-laminA/C (636) (sc-7292, 1:500, Santa Cruz Biotechnology), anti-phospho-Ser22LaminA (2026, 1:200, Cell Signaling), anti-CDK4 (DC5156, 2906, 1:100, Cell Signaling), anti-CDK2 (M-2, SC-163, 1:200, Santa Cruz Biotechnology), anti-CDK6 (C-21, SC-177, 1:200, Santa Cruz Biotechnology), anti-p38 (9212, 1:200, Cell Signaling), anti-phospho-MSK1 (9595, 1:200, Cell Signaling), anti-phospho-RSK (9341, 1:200, Cell Signaling), anti-MEK1/2 (9122, 1:200, Cell Signaling), anti-phospho-ERK1/2 (4370, 1:400, Cell Signaling) diluted in PBS/BSA. Next, cells were washed three times in PBS/BSA and incubated with the appropriate secondary antibody (1:2000, AlexaFluor 488 goat anti-mouse, AlexaFluor 568 goat anti-mouse, or AlexaFluor 488 goat anti-rabbit, Molecular Probes-Thermofisher-Scientific) for 1h at room temperature. Finally, cells were rinsed three times with PBS alone and once with PBS containing 300 nM DAPI for 10 minutes. Images from independent fields were captured with a fluorescence microscope and processed with Metamorph.

For superresolution structured illumination microscopy, cells were fixed as described above and stained with anti-lamin A/C antibody (as above) or anti-fibrillarin (C13C3, 2639, Cell Signaling Technology, 1:200). After washing they were stained with Alexa Fluor 568 goat anti-rabbit IgG (H+L) (A11029, Life Technologies, 1:2000) and Alexa Fluor 568 goat anti-rabbit IgG (H+L) (A11036, Life Technologies, 1:2000). Images were obtained with Super Resolution microscope Axio Observer Z1 ZEISS Elyra PS.1, SR-SIM, 3D PALM, STORM from Zeiss. We picked up images sets of 5 subsets each captured after rotating the grid by 5 degrees. A high resolution image was extracted from the raw data using the Structured Illumination Microscopy and the Maximum Intensity Projection algorithms at a lateral resolution (XY) of 120 nm and an axial resolution (Z) of 300 nm.