Phosphorylation at serine 482 affects stability of NF90 and its functional role in mitosis

Abstract

Objectives:  NF90 is a multifunctional double‐strand RNA binding protein with documented roles in transcription, mRNA stability, translation, RNA processing and transport, and mitosis. It is a phosphoprotein that interacts with, and is a substrate for, several protein kinases. The study described here was initiated to gain better understanding of specific NF90 phosphorylation sites and their relationship to mechanisms by which NF90 performs its various functions.

Materials and methods:  Phosphoproteomic studies have identified NF90 serine 482 (S482) as a major phosphorylation site in vivo. Site‐specific mutations were introduced at this site and the mutated proteins were expressed in MCF7 cells by transfection. Western blotting was used to examine NF90 expression, stability, and responsiveness to protein kinase activators and inhibitors. Flow cytometry was used to examine effects of NF90 mutation on cell cycle progression.

Results:  Non‐phosphorylatable mutant S482A was unstable compared to phosphomimetic S482E mutant. NF90‐S482A expression was greatly enhanced by inhibiting proteasomal degradation or by activating PKC. Identical treatments had little effect on NF90‐S482E. In contrast to WT NF90 or NF90‐S482E, cells stably expressing NF90‐S482A accumulated in M phase when treated with TPA.

Conclusions:  Phosphorylation at S482 is important for NF90 stability and in regulating its functional role during mitosis. Based on the sequence surrounding S482, mitotic kinase PLK1 is a strong candidate for the enzyme that phosphorylates NF90 at this site.

Introduction

NF90 is an RNA‐binding protein containing two double‐stranded RNA binding motifs (dsRBMs) in the C‐terminal half of the molecule. NF90 and its various isoforms, the most prominent of which is called NF110, are derived from the same gene, ILF3, through alternative splicing (1, 2). Other names for NF90 include interleukin enhancer binding factor 3 (ILF3) (1, 3), human translational control protein 80 (TCP80) (4), M phase phosphoprotein 4 (MPP4) (5), double‐strand RNA binding protein 76 (DRBP76) (6) and nuclear factor associated with dsRNA (NFAR1) (2). Multiple names for this factor exist because it was identified through several different approaches and found to have multiple cellular functions.

The first functional characterization of NF90 showed that it acts as a transcription factor by binding to the promoter of IL‐2 gene (7, 8); subsequently, several other genes have been reported to be regulated by NF90. These include HLA‐DR alpha (9) and interleukin‐13 genes (10). NF90 appears to be a component of a large transcriptional control complex where it interacts with other proteins such as NF45, Ku70 and Ku80 (11). Depending on promoter context, NF90 can act as either a positive or as a negative regulator of gene expression (12). NF90 also inhibits transcription of HIV genes by binding to TAR RNA and inhibition of Tat‐transactivation of HIV‐1 LTR (13).

Further studies have provided evidence that NF90 is involved in translational control, mRNA stability, viral replication, mRNA and microRNA processing, and mitosis. Translational control was first demonstrated for β‐glucosidase mRNA in which NF90 binds to the coding region to inhibit synthesis of the protein (4). In contrast, stability or translation of a number of mRNAs has been shown to be enhanced by NF90 binding to the 3′‐UTR. This activity is generally attributed to binding to specific AU‐rich motifs in target mRNAs, which include those that encode IL‐2 (14), p21Cip1 (15), VEGF (16) and MKP‐1 (17); NF90 may also bind and stabilize its own mRNA 3′‐UTR (18). Recently, Kuwano et al. (19) identified a large number of mRNAs that interact with NF90. They characterized an AU‐rich NF90 signature motif in the 3′‐UTRs of many of these mRNAs and found that NF90 repressed translation through this element.

Other cell functions of NF90 are less well characterized. Parrott and Mathews (20) identified a novel family of small NF90‐associated RNAs (snaRs); these are highly structured non‐coding RNAs abundantly expressed in some human tissues. The function of these RNAs remains unknown, but it is thought that they may modulate expression of nearby genes through epigenetic mechanisms (20). NF90 has also been shown to interact with primary, unprocessed microRNAs (21) and this interaction inhibits biogenesis of mature miRNAs, possibly by blocking access of the microprocessor complex to primary miRNAs transcripts. Finally, there is evidence that NF90 plays an important role in mitosis, it has been identified as an antigen for the MPM2 antibody, which is reactive with phosphoproteins that are abundant in mitosis (5). Phosphorylation of NF90 at MPM2 recognition sites is associated with its translocation to the cytosol at the onset of mitosis (22). Recently, this same group showed that repression of either NF90 or its binding partner, NF45, leads to defective mitosis and accumulation of multinucleate giant cells (23).

It is apparent that NF90 is a multifunctional protein, but the mechanisms by which it performs its various roles are not well understood. It is also not well understood how NF90 activity is regulated with respect to each of its functions. Several protein–protein interactions have been identified and these may confer specific functions to NF90 (2, 6, 11, 24, 25, 26, 27). However, phosphorylation appears to be a major contributor to regulation of various activities of NF90. As mentioned above, NF90 is highly phosphorylated during mitosis at sites that are recognized by MPM2 antibody (5, 22). Early studies also showed that phosphorylation is important for NF90 binding to elements in the IL‐2 promoter (7); also, NF90 may be a substrate for several different kinases. MPM2 antibody recognizes proline‐directed phosphorylation sites, suggesting that NF90 may be a substrate for cyclin‐dependent kinases (CDKs) or mitogen‐activated kinase (MAPK) families. NF90 interacts with, and is a substrate for both PKR (6, 25, 26) and DNA‐PK (11, 24). Xu and Grabowski (28) showed that inhibition of protein kinase C (PKC) correlates with reduction in NF90 phosphorylation. They suggested that NF90 may be a direct target of PKC as there are multiple potential target sites for this enzyme in NF90. Recently, Pei et al. (29) demonstrated that NF90 is phosphorylated by AKT at serine 647 and that this is associated with nuclear export and stabilization of IL‐2 mRNA.

Although phosphorylation appears to play a key role in regulating NF90, biological consequences of phosphorylation and specific amino acids involved are, for the most part, unknown. Several recent phosphoproteomic studies have identified a number of sites in NF90 that are phosphorylated in vivo (30, 31, 32, 33) and we have begun to examine these various sites through site‐specific mutagenesis. Data presented here indicate that phosphorylation of NF90 at serine 482 is involved in stabilizing the protein and in regulating its functional role during mitosis.

Materials and methods

Cell culture

Human breast cancer cell line MCF7 and human embryonic kidney cell line HEK‐293T were purchased from American Type Culture Collection (Rockville, MD, USA). The cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Atlanta Biologicals) in a humidified incubator at 37 °C with 5% CO₂.

Chemicals and reagents

Dimethyl sulphoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), nocodazole, propidium iodide, leupeptin and aprotinin were purchased from Sigma (St Louis, MO, USA). Gö6976, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), PD98059, LY294002, PKR inhibitor (PKRI), and MG132 were purchased from Calbiochem (La Jolla, CA, USA). Final concentrations are indicated in each figure. Dithiothreitol and G418 sulphate were purchased from Fisher Scientific (Pittsburgh, PA, USA). Phenylmethylsulfonyl fluoride was purchased from Pierce (Rockford, IL, USA). Lipofectamine, Plus reagent, Lipofectamine 2000 and aprotinin were purchased from Invitrogen (Carlsbad, CA, USA). DreamFect was purchased from Boca Scientific, Boca Raton, FL, USA). Sodium dodecyl sulphate (SDS) was purchased from Bio‐Rad Laboratories (Hercules, CA, USA). Restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA) or Promega (Madison, WI, USA).

Antibodies

Indicated dilutions were used for western blot analysis. Anti‐DRBP76 (anti‐NF90, 1:1000) mouse monoclonal antibody was purchased from BD Biosciences (San Diego, CA, USA). Anti‐Xpress mouse monoclonal antibody (1:5000) was purchased from Invitrogen. MPM‐2 mouse monoclonal antibody (1:1000) was purchased from Millipore (Billerica, MA, USA). Anti‐GAPDH mouse monoclonal antibody (1:1000) and anti‐β‐actin mouse monoclonal antibody (1:10 000) were purchased from Sigma. HRP‐conjugated goat anti‐mouse and goat anti‐rabbit IgG secondary antibodies (1:10 000) were purchased from Thermo Scientific (Waltham, MA, USA).

Site‐directed mutagenesis

pcDNA3.1/HisB‐NF90a (WT NF90) was a gift from Dr Michael Mathews (Department of Biochemistry and Molecular Biology, New Jersey Medical School). This construct was used as template to generate mutations in this study with a 3‐step PCR method. The first PCR paired primers NF90His1 and the mutant 3′ primer and the second PCR paired primers NF90His2 and the mutant 5′ primer. In the third PCR, PCR products of the first two steps were combined following extraction of fragments from an agarose gel and used as template. 5′ primer NF90His1 and 3′ primer NF90His2 were used as primers for the third PCR reaction. PCR products were digested with EcoR I and Hind III, purified on a Tris‐acetate EDTA agarose gel and then ligated into vector pcDNA3.1/HisB digested with EcoR I and Hind III. Ligation products were used to transform Escherichia coli (NEB 5α) competent cells. Transformed E. coli were plated on TB agar plates containing ampicillin and plasmids obtained from colonies were sequenced to confirm the mutations. Sequences of primers used for site‐directed mutagenesis are available upon request.

Transient and stable transfection

MCF7 cells were plated in 35 mm dishes. After overnight incubation, cells were transfected with Xpress‐tagged NF90 constructs using Lipofectamine Plus reagent. For transient transfection, cells were harvested 24 h after transfection for western blot analysis. For stable transfection, cells were transferred to 100 mm dishes after incubating at 37 °C for 24 h. G418 (1 mg/ml) was used to select for colonies of stably transfected cells. Individual colonies were then picked and expanded in DMEM supplemented with 10% foetal bovine serum and G418. Stable transfection was confirmed by western blotting with Xpress antibody.

Western blot analysis

Cells in 35 mm dishes were rinsed once with 1× phosphate‐buffered saline (PBS) and lysed with 150 μl of 1× sodium dodecyl sulphate (SDS) sample buffer [2.5 mm Tris–HCl (pH 6.8), 2.5% SDS, 100 mm DTT, 10% glycerol and 0.025% pyronine Y]. Equal amounts of proteins from each sample were fractionated by SDS polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Immobilin P, Millipore) using a Bio‐Rad Trans‐blot SD semi‐dry electrophoretic transfer cell in a transfer buffer containing 48 mm Tris–HCl and 39 mm glycine. Membranes were blocked with 5% (w/v) non‐fat dry milk in Tris‐buffered saline [TBS, 10 mm Tris‐Cl (pH 7.5), and 150 mm NaCl] containing 0.1% Tween‐20 (TBS‐T) for 15–60 min at room temperature. Blots were probed with appropriate dilutions of primary antibody in TBS‐T containing 5% non‐fat dry milk, for 1 h at room temperature or overnight at 4 °C. After washing in TBS‐T for 1 h, membranes were probed with horseradish peroxidase (HRP)‐conjugated anti‐mouse or anti‐rabbit IgG secondary antibody. After washing in TBS‐T for 30 min, membranes were incubated in Super Signal West Pico chemiluminescent substrate (Thermo Scientific) and bands were then visualized by exposure to film.

Flow cytometry

Cells (1 × 10⁶) were plated in duplicate 60 mm dishes and incubated overnight followed by treatment with DMSO or TPA (20 ng/ml) for 24 h. They were then trypsinized and trypsin was inactivated with 1× PBS containing 10% FBS. Cells were pelleted at ∼800 ×g 5 min at room temperature using a Sorvall T6000D benchtop centrifuge. They were then resuspended in 2 ml of cold 70% ethanol and fixed at −20 °C overnight. Fixed cells were pelleted and stained with 1 ml of Telford reagent [33.62 μg/ml EDTA, 26.8 μg/ml RNase A, 0.05 mg/ml propidium iodide, 0.1% Triton X‐100 in PBS] and were then filtered into 5 ml polystyrene round‐bottom tubes using a 0.2 μm mesh. Ten thousand events were collected from each tube with a FACSVantage SE flow cytometer and data were analysed using cellquest and modfit software.

Results

Mass spectrometry has been developed as a tool for identifying phosphorylation sites across the entire proteome of living cells. Several such studies have identified phospho‐peptides derived from NF90 (30, 31, 32, 33), most of which have been identified in two or more of the studies (Table 1), suggesting that they are authentic phosphorylation sites in vivo. Using recently developed methods, Dephoure et al. (32) were able to measure relative abundance of phospho‐peptides in cells arrested in either M phase or G₁. For NF90, the peptide containing phosphorylated serine 482 (S482) was the most abundant phospho‐peptide and this site was highly phosphorylated in both M and G₁ phases. S482 was also identified as a phosphorylation site in all other phosphoproteomics studies referenced above (see Table 1). This amino acid is located in the region linking the two dsRBMs (Fig. 1a) and it seems likely that phosphorylation at this site could influence NF90 function. To address this possibility, site‐specific mutagenesis was used to convert S482 to alanine (S482A) or to glutamate (S482E). The S482A mutant cannot be phosphorylated while S482E mutant should act as a phosphomimetic substitution.

Table 1.

 NF90 phosphopeptides identified by phosphoproteomics

Study	Phospho‐AA	Peptide sequence
Beausoleil et al. (30)	S482	DSSKGEDS*AEETEAKPAVVAPAPV VEAVSTPSAAFPSDATAEQGPILTK
	S62	GSSEQAES*DNMDVPPEDDSK
	T592	LFPDT*PLALDANK
Van Hoof et al. (33)	T368	PSTTYATT*PMKRPME
	S476	TGAEGRDS*SKGEDSA
	S477	GAEGRDSS*KGEDSAE
	S482	DSSKGEDS*AEETEAK
	T486	GEDSAEET*EAKPAVV
	T592	LEKLFPDT*PLALDAN
Chen et al. (31)	S476	TGAEGRDS*SKGEDSA
	S477	GAEGRDSS*KGEDSAE
	S482	DSSKGEDS*AEETEAK
	T486	GEDSAEET*EAKPAVV
	T504	PVVEAVST*PSAAFPS
	T592	LEKLFPDT*PLALDAN
Dephoure et al. (32)	S57	DEQRKGSS*EQAESDN
	S62	GSSEQAES*DNMDVPP
	S190	VLAGETLS*VNDPPDV
	T364	VQIPPSTT*YAITPMK
	Y365	QIPPSTTY*AITPMKR
	T368	PSTTYAIT*PMKRPME
	S382	EEDGEEKS*PSKKKKK
	T469	LQDMGLPT*GAEGRDS
	S476	TGAEGRDS*SKGEDSA
	S477	GAEGRDSS*KGEDSAE
	S482	DSSKGEDS*AEETEAK
	T486	GEDSAEET*EAKPAVV
	S503	APVVEAVS*TPSAAFP
	T504	PVVEAVST*PSAAFPS
	Y579	NKKVAKAY*AALAALE
	T592	LEKLFPDT*PLALDAN

References for all studies are indicated in the first column. Number of putative phosphorylated amino acids is indicated in the middle column with S482 shown in bold. The right column indicates sequences of identified phosphopeptide with an asterisk following the phosphorylated amino acid.

Figure 1.

  Mutation of NF90 S482 affects protein stability. (a) Diagram of NF90 showing major domains, position of S482 between the two dsRBMs is shown. (b) Left panel: Constructs encoding WT NF90 and mutant NF90, in which S482 was converted to either alanine (A) or glutamate (E) were transiently transfected into MCF7 cells. Xpress‐tagged proteins were detected by western blotting. NS represents an endogenous protein that is non‐specifically recognized by Xpress antibody and is shown as a loading control; control cells (Con) were transfected with empty vector. Numbers in parentheses below each lane represent relative band intensities normalized to the NS band. Right panel: Extracts from MCF7 cells stably expressing WT NF90, NF90‐S482A, or NF90‐S482E were used to detect NF45 by western blotting. β‐actin is shown as loading control. (c) MCF7 cells stably expressing WT NF90, NF90‐S482A, or NF90‐S482E were treated with vehicle (DMSO) or proteasomal inhibitor MG132 (10 μm) for 24 h. Total cell lysates were prepared and used for western blotting with Xpress antibody to detect recombinant proteins or anti‐DRBP76 to detect endogenous NF90 and NF110. Numbers in parentheses below each lane represent relative band intensities normalized to the actin band.

Expression constructs encoding these mutant NF90 constructs, as well as WT NF90 protein, were transiently transfected into MCF7 cells. Expressed proteins were detected by western blotting for the Xpress epitope tag located at the N terminus of the recombinant proteins (Fig. 1b). Surprisingly, NF90‐S482E was expressed at much higher levels than either NF90‐S482A or the WT protein, the only difference between these expression constructs being a single amino acid codon. It is therefore unlikely that the observed variation in expression of NF90 mutants is due to differences in efficiency of transfection, transcription of constructs, or translation of encoded proteins; it is more likely that these results indicate that phosphorylation of NF90 at S482 affects protein stability. To examine this possibility, MCF7 cells stably expressing Xpress‐tagged WT NF90, NF90‐S482A, or NF90‐S482E were treated with proteasome inhibitor MG132 (Fig. 1c). Levels of both WT and S482A mutant were considerably increased following MG132 treatment. In absence of the inhibitor, phosphomimetic S482E mutant was expressed at higher levels than either WT NF90 or NF90‐S482A and showed very little change in presence of MG132. These results suggest that NF90 is degraded by the proteasome and that phosphorylation at S482 inhibits turnover of NF90 through this pathway. Interestingly, levels of endogenous NF90 did not appear to be altered by proteasome inhibition.

NF90 forms a heterodimer with NF45, and NF45 has been shown to influence stability of NF90 (23), however, NF45 is expressed at similar levels in cells expressing WT NF90, NF90‐S482A and NF90‐S482E (Fig. 1b). Thus, differences in expression levels of NF90 mutants cannot be explained by altered NF45 expression.

To investigate further the signalling pathways that may contribute to NF90 stability, we treated stably transfected MCF7 cells with specific activators or inhibitors. When cells were treated with TPA, an activator of PKC, there was a substantial increase in NF90‐S482A levels (Fig. 2a). Levels of the S482A mutant also decreased somewhat after treatment with Gö6976, a PKC inhibitor (Fig. 2a,b). Inhibitors of ERK (PD98059) and JNK (SP600125) pathways modestly decreased levels of NF90‐S482A expression, while inhibitors of PKR and PI3K had no effect (Fig. 2a). The increase in NF90‐S482A levels in cells treated with TPA was observed as early as 4 h after treatment started and continued to increase up to 24 h (Fig. 2c). The striking effect of TPA on the NF90‐S482A mutant was not observed for either WT NF90 or NF90‐S482E mutant in stably transfected MCF7 cells (Fig. 2b). NF90‐S482E was already expressed at high levels in the absence of TPA, thus, it appears that NF90 is less stable when S482 is unphosphorylated and that activation of PKC impedes turnover of this form of NF90.

Figure 2.

  TPA enhances expression of NF90‐S482A in stably transfected MCF7 cells. (a) MCF7 cells stably transfected with NF90S‐482A were treated with indicated inhibitors and activators for 24 h. Levels of NF90‐S482A were determined by western blotting using Xpress antibody. β‐actin was detected as loading control. (b) MCF7 cells stably expressing WT NF90, NF90‐S482A, or NF90‐S482E were treated with PKC activator TPA (10 ng/ml) or PKC inhibitor Gö6976 (10 μm) for 24 h. Western blots were performed as in (a). (c) Time course of TPA induction of NF90‐S482A. MCF7 cells stably expressing either NF90‐S482A or NF90‐S482E were treated with TPA (20 ng/ml). Cells were harvested at 0, 2, 4, 6, 12 and 24 h time points following addition of TPA to the medium. Western blotting was used to detect exogenous NF90 proteins (Xpress) or β‐actin. Numbers in parentheses below each lane represent relative band intensities normalized to actin levels.

Guan et al. (23) recently showed that NF90, together with its binding partner NF45, plays a critical role in cell division through its involvement in mitotic control. In addition, NF90 is known to be highly phosphorylated in mitosis and work of Dephoure et al. (32) indicates that S482 is one of the most highly phosphorylated sites in NF90 during mitosis. These findings suggest that S482 phosphorylation plays a role in controlling mitotic function of NF90. To test this, MCF7 cells stably transfected with constructs encoding WT NF90, NF90‐S482A, or NF90‐482E were treated with vehicle (DMSO) or TPA for 24 h and then analysed by flow cytometry, to determine percentages of cells in each phase of the cell cycle (Fig. 3). Overall, in the absence of TPA, cells expressing NF90‐S482A and NF90‐S482E were similar to cells expressing WT NF90; there was a small increase in G₂/M phase cells with NF90‐S482A. For NF90‐S482E, there was an increase in G₁ phase cells with corresponding decrease in S phase cells. Addition of TPA to the cultures led to accumulation of cells expressing WT NF90 and NF90‐S482E in G₁, with parallel decrease in S phase cells. When cells expressing NF90‐S482A were treated with TPA, there was also a decrease in S phase cells, but cells accumulated in G₂/M rather than in G₁. These results indicate that phosphorylation at S482 is important for NF90 function in mitosis.

Figure 3.

  Mutation of NF90 at S482 affects mitosis. MCF7 cells stably expressing WT NF90, NF90‐S482A, or NF90‐482E were treated with DMSO (left‐hand panels) or with TPA (10 ng/ml, right‐hand panels) for 24 h. Cells were fixed, stained with propidium iodide and analysed by flow cytometry. ModFit software was used to estimate percentages of cells in G₁, S, or G₂/M phases, as indicated in each panel.

During mitosis, NF90 becomes highly phosphorylated at sites that are recognized by MPM2 antibody (5, 22). It is possible that S482 represents an MPM2 recognition site and that this is the reason that S482A mutant affects NF90 function in mitosis; this is unlikely though, because MPM2 is thought to recognize proline‐directed phosphorylation sites (34). However, phospho‐S482 could influence phosphorylation at MPM2 sites if, for example, it acts as a priming site to recruit a proline‐directed kinase such as the M phase kinase CDK1. To determine whether S482A mutant alters MPM2 phosphorylation, WT NF90 and NF90‐S482A constructs were transiently transfected into HEK 293t cells. Cells were treated with nocodazole for 24 h to arrest them in M phase and then western blotting was performed using MPM2 antibody or Xpress antibody to detect recombinant NF90 (Fig. 4). NF90‐S482A was expressed at lower levels than WT NF90 as observed previously; however, NF90‐S482A was still highly phosphorylated at sites recognized by MPM2 antibody. Thus, phosphorylation at MPM2 sites is not dependent on phosphorylation at S482.

Figure 4.

  Mutation of NF90 at S482 does not affect phosphorylation at MPM2 recognition sites. HEK‐293T cells were transiently transfected with expression constructs encoding either WT NF90 or NF90‐S482A. Control cells were transfected with empty pcDNA3.1/HisB vector (Con). Twenty‐four hours after transfection, cells were treated with nocodazole (0.15 μg/ml) for 24 h to arrest cells in M phase. Whole cell lysates were analysed by western blotting using MPM‐2 or Xpress antibody. Numbers in parentheses below the lanes represent relative intensities of the rNF90 bands detected with the MPM2 antibodies.

Discussion

Phosphorylation appears to be a key mechanism for regulation of NF90 activity and localization within the cell. However, little is known about specific phosphorylation sites, functional roles of the various sites, or protein kinases involved in phosphorylating them. Several phosphoproteomic studies have identified NF90 amino acids that are phosphorylated in vivo (30, 31, 32, 33); all these studies identified S482 as a phosphorylation site. The quantitative study carried out by Dephoure et al. (32) indicates that this amino acid is highly phosphorylated in both G₁ and M phases and the work presented here indicates that S482 phosphorylation is important for NF90 stability and for its functional role in mitosis.

Endogenous NF90 is known to have a relatively long half‐life of several days, this is dependent on its interaction with its heterodimerization partner, NF45 (23). When NF45 expression is repressed by siRNAs, NF90 expression is dramatically reduced. Likewise, when NF90 is depleted, NF45 becomes unstable. Thus, endogenous NF90 expression is balanced with endogenous NF45 expression. Based on these findings, it is expected that endogenous NF90, which is stabilized by NF45, would not be significantly affected by proteasome inhibition. However, ectopically expressed NF90, above levels of endogenous NF45, would be expected to be rapidly turned over and be stabilized by proteasome inhibition. This notion is supported by the data presented in Fig. 1c.

When S482 is mutated to the non‐phosphorylatable amino acid alanine, NF90 is expressed at much lower levels than when it is mutated to the phosphomimetic amino acid glutamate. This appears to be because of enhanced turnover of NF90‐S480A mutant, since inhibition of proteasome activity restored expression to levels comparable to NF90‐S480E. There are several possibilities for this finding. One is that phosphorylation at S482 enhances binding of NF90 to NF45 and that NF90‐S482A is less stable as it does not bind efficiently to NF45. Another possibility is that phosphorylation at S482 inhibits targeting of the protein for degradation by the proteasome. Phosphorylation of S482 could also affect protein–protein interactions, RNA binding, or subcellular localization of NF90, all of which could affect stability of the protein. Further experiments will be required to determine the mechanism by which S482 phosphorylation influences NF90 expression levels.

Expression of NF90‐S482A is greatly enhanced by treating cells with TPA and reduced by treating with Gö6976, suggesting that these effects are through PKC. Xu and Grabowski (28) have previously reported that inhibition of PKC correlates with reduction of NF90 phosphorylation in HepG2 cells. They found 10 potential PKC target sites by comparing the sequence of NF90 with PKC consensus sequence. Two potential PKC target sites, S382 and S476, were identified by phosphoproteomic methods as being phosphorylated in vivo (32). S476 is close to S482 in the region between the two dsRBMs; it is possible that phosphorylation at either site enhances NF90 stability. This is suggested by the finding that phosphomimetic mutant NF90‐S482E is highly stable and not responsive to TPA, whereas NF90‐S482A mutant is unstable and highly responsive to TPA. The S482A mutation appears to unmask effects of phosphorylation by PKC, possibly at S476, on NF90 stability. Wild‐type NF90 can be phosphorylated at either S482 site or the PKC target site; and phosphorylation at either site may stabilize NF90. However, it is clear that the S482E mutant is more stable and expressed at higher levels than wild‐type protein in the presence of TPA (see Fig. 2). There are several possibilities that could explain this. The wild‐type protein may exist in a state where it is phosphorylated at the S482 site and not at the PKC site, at the PKC site and not at S482 site, at both sites, or at neither site. Phosphorylation status is likely to be dependent on the state of the cell and phase of the cell cycle. Our data would suggest that only NF90 that is not phosphorylated at either S482 site or at the PKC target site would be responsive to TPA, in terms of protein stability – this pool of NF90 may be small in logarithmically growing, asynchronous cell populations. Also, even though phosphorylation at either S482 site or the PKC target site can lead to stabilization of NF90, the two sites cannot be considered equivalent. Each site may differentially influence protein–protein interactions or subcellular localization, or serve as a priming site for further modifications. All of these could potentially influence expression levels and stability of the protein. Distinguishing these various possibilities will require definitive identification of the PKC site that mediates protein stabilization, creating single and double mutations, and analysis in quiescent cells and cells synchronized at various stages of the cell cycle.

MCF7 cells have previously been shown to undergo cell cycle arrest in G₀/G₁ when treated with TPA (35, 36, 37). This is associated with up‐regulation of p21CIP1 and decreased phosphorylation of Rb (38, 39). MCF7 cells expressing wild‐type NF90 or NF90‐S482E also arrest in G₀/G₁ when treated with TPA. In contrast, there is accumulation of cells with G₂/M DNA content following TPA treatment of cells expressing NF90‐S482A. These results suggest that phosphorylation of NF90 at S482 is important for mitosis and that non‐phosphorylatable NF90‐S482A acts in a dominant negative manner. As it is expressed at low levels in the absence of TPA, NF90‐S482A generally has minimal effect on cell cycle progression; however, when induced to high levels by treating cells with TPA, NF90‐S482A interferes with the process of mitosis. MCF7 cells expressing NF90‐S482E retain mitotic function of wild‐type NF90 and pass through mitosis and arrest in G₀/G₁ like the parental cell line.

A further question that arises from the current work concerns the identity of the kinase that phosphorylates S482; a strong possibility is polo‐like kinase 1 (PLK1). The consensus substrate specificity for PLK1 was first defined by Nakajima et al. (40) as E/D‐X‐pS/T‐Φ‐D/E, where X is any amino acid and Φ is any hydrophobic amino acid. Ahonen et al. (41) and Dephoure et al. (32) have defined very similar consensus substrate sites for PLK1. The sequence surrounding S482, G‐E‐D‐pS‐A‐E‐E, is a very strong match for the PLK1 consensus substrate sequence. A known in vivo substrate of PLK1, Wee1, is phosphorylated within the sequence G‐E‐D‐pS‐A‐F‐Q (42), which is very similar to the region surrounding S482. PLK1 is known to play an essential role in entry into and progression through mitosis (42). PLK1 is targeted to substrates through its polo‐box domain (PBD) which interacts with phosphorylated peptides (43). Docking sites for PLK1 include proline‐directed phosphorylation sites and it binds to some MPM2 recognition sites that have core sequence S‐pT‐P (44). One potential MPM2 recognition site in NF90 is at T504 and is within the sequence V‐S‐pT‐P S, which is likely to be recognized by the PLK1 PBD. It can therefore be speculated that NF90 is first phosphorylated at T504 and that this serves as a docking site for PLK1, which subsequently phosphorylates S482. This remains to be confirmed by direct experimentation.