Regulation of microtubule dynamics through phosphorylation on stathmin by Epstein-Barr virus kinase BGLF4

Abstract

Stathmin is an important microtubule (MT)-destabilizing protein, and its activity is differently attenuated by phosphorylation at one or more of its four phosphorylatable serine residues (Ser-16, Ser-25, Ser-38, and Ser-63). This phosphorylation of stathmin plays important roles in mitotic spindle formation. We observed increasing levels of phosphorylated stathmin in Epstein-Barr virus (EBV)-harboring lymphoblastoid cell lines (LCLs) and nasopharyngeal carcinoma (NPC) cell lines during the EBV lytic cycle. These suggest that EBV lytic products may be involved in the regulation of stathmin phosphorylation. BGLF4 is an EBV-encoded kinase and has similar kinase activity to cdc2, an important kinase that phosphorylates serine residues 25 and 38 of stathmin during mitosis. Using an siRNA approach, we demonstrated that BGLF4 contributes to the phosphorylation of stathmin in EBV-harboring NPC. Moreover, we confirmed that BGLF4 interacts with and phosphorylates stathmin using an in vitro kinase assay and an in vivo two-dimensional electrophoresis assay. Interestingly, unlike cdc2, BGLF4 was shown to phosphorylate non-proline directed serine residues of stathmin (Ser-16) and it mediated phosphorylation of stathmin predominantly at serines 16, 25, and 38, indicating that BGLF4 can down-regulate the activity of stathmin. Finally, we demonstrated that the pattern of MT organization was changed in BGLF4-expressing cells, possibly through phosphorylation of stathmin. In conclusion, we have shown that a viral Ser/Thr kinase can directly modulate the activity of stathmin and this contributes to alteration of cellular MT dynamics and then may modulate the associated cellular processes.

FIGURE 1.

Increasing phosphorylation of stathmin in LCL with spontaneous lytic cycle progression. A, cell lysates were harvested from EBV-transformed LCL cell lines (P1, P7, P9, P13, P14, and P15) and subjected to immunoblotting analysis. Cell lysates were probed by specific antibodies against EBNA2, BGLF4, Zta, phosphorylated stathmin at serine-16, nonphosphorylated stathmin, and β-actin. The band densities of nonphosphorylated and total phosphorylated stathmin of each LCL (P13, P14, P1, and P15) relative to the P7 were quantified and normalized to the intensity of the corresponding internal control using ImageQuant software. These densities are indicated below the corresponding bands. B, P1, P7, and P13 LCL were stained with anti-BGLF4 (red color) and anti-phosphorylated stathmin (green color) antibodies. Nuclear DNA was stained with Hoechst 33258. BGLF4-expressing cells are indicated by arrows. C, an EBV-positive nasopharyngeal cell line, NA, was co-transfected with pSG5-Rta and si-BGLF4-1, si-BGLF4-2 or control siRNA fragments. Cell lysates were harvested 36 h post-transfection and subjected to immunoblotting. The band densities of nonphosphorylated and total phosphorylated stathmin of each transfection relative to the vector and si-CTR transfection (lane 1) were quantified and normalized to the intensity of the corresponding internal control using ImageQuant software. These densities are indicated below the corresponding panels, and relative band densities of phosphorylated stathmin from three independent experiments are statistically analyzed and plotted. *, significant difference from vector control (p < 0.05).

FIGURE 2.

Increasing phosphorylation of stathmin in BGLF4-expressing 293 cells. A and B, doxycycline-driven BGLF4 or BGLF4 kinase dead mutant (BGLF4-KD) expressing 293 cells were induced by 10 ng/ml doxycycline for indicated times (induction: +; non-induction: −). C and D, HeLa cells were transfected with various amounts of BGLF4- or BGLF4-KD-expressing plasmids and then lysates were harvested at 24 h post-transfection. Cell lysates were analyzed by immunoblotting and proteins of interest were detected by specific antibodies against BGLF4, nonphosphorylated stathmin, phosphoserine 16 stathmin, and GAPDH. Positive controls of hyperphosphorylated stathmin were harvested from Jurkat or HeLa cells treated with nocodazole (NOC). The same blot was stripped and then re-probed with anti-C-terminal stathmin antibody. Hyperphosphorylated forms of stathmin are indicated by the asterisks in C.

FIGURE 3.

Stathmin is phosphorylated by BGLF4 in vitro. A, recombinant GST-stathmin, GST control protein, and a positive control protein, Histone H1, were used as substrates. HeLa cells were transfected with BGLF4 or BGLF4-kinase dead expression plasmids and BGLF4 (K) and its kinase dead mutant (KD) were obtained by immunoprecipitation with anti-BGLF4 antibody from total cell lysates 24 h post-transfection. IP kinase assays were carried out for 30 min as described under “Experimental Procedures.” The amounts of substrates and kinase loaded are shown. The experiment was performed twice, and a representative example is shown. BGLF4 was observed to phosphorylate GST-stathmin and Histone H1 protein but not GST control protein. B, positions of serine phosphorylation sites in wild-type stathmin and purified stathmin mutants are shown. Phosphorylatable residues of GST-tagged stathmin mutants, which can only be phosphorylated at one of four target residues in the N terminus of stathmin, such as SAAA (only phosphorylatable on residue 16), ASAA (only phosphorylatable on residue 25), AASA (only phosphorylatable on residue 38) and AAAS (only phosphorylatable on residue 63) are indicated. C, substrates of GST-tagged stathmin mutants in B were used in the same reaction as described in A. BGLF4 can be seen to phosphorylate GST-stathmin mutants mainly at residues 16, 25, and 38. The experiment was performed twice, and a representative example is shown.

FIGURE 4.

BGLF4 interacts with stathmin and induces phosphorylation of stathmin in vivo. A, HeLa cells were transiently transfected with either pSG5-BGLF4 or pSG5-BGLF4 combined with pSG5-stathmin-Flag-expressing plasmids. Cell lysates were harvested 18 h post-transfection and immunoprecipitated by anti-BGLF4, anti-Flag antibodies, or mouse IgG antibodies. Immunoprecipitates were subjected to immunoblotting and then probed with anti-BGLF4, anti-Flag, or anti-stathmin antibodies. The Flag-tagged stathmin is indicated by the asterisk. B, inducible BGLF4, BGLF4-KD (BGLF4 kinase dead form), or vector control 293 T-REx cells were induced with 10 ng/ml doxycycline for 24 h. Lysates were subjected to two-dimensional PAGE and probed with anti-stathmin or anti-phosphorylated Ser-16 stathmin antibodies. N, nonphosphorylated form of stathmin. P1, stathmin isoforms which are phosphorylated at one phosphorylatable residue (dot 1); P2, stathmin isoforms which are phosphorylated at two phosphorylatable residues (dots 2 and 3); P3, stathmin isoforms which are phosphorylated at three phosphorylatable residues (dots 4 and 5). C, ratio for relatively density of hyperphosphorylated stathmin (dots 4 and 5) and hypophosphorylated stathmin isoforms (dots 1, 2, 3, 6, and 7) from the two experiments are plotted and statistically analyzed. *, significant difference to vector control (p < 0.05).

FIGURE 5.

Microtubule networks are altered in cells expressing BGLF4, stathmin-4E (pseudo-phosphorylated stathmin), wild-type stathmin, and stathmin-4A (nonphosphorylatable stathmin). Slide-cultured HeLa cells were transfected with vector, BGLF4, BGLF4-KD, stathmin-4E, stathmin, or stathmin-4A expression plasmids. Twenty-four hours post-transfection, cells were fixed in 4% paraformaldehyde and stained with anti-BGLF4 Ab, anti-β tubulin Ab, anti-Flag Ab, and Hoechst 33258. Microtubule networks can be categorized by normal, diffuse, or disorganized types in vector- (A), BGLF4- (B), BGLF4-KD- (C), stathmin-4E- (D), stathmin- (E), and stathmin-4A-expressing cells (F). G, variations of microtubule networks observed in different transfections are calculated from two independent experiments and more than 200 cells were counted in each transfection. The counts of each categorical variable (disorganized, diffuse, or normal type of MT) in one treatment (BGLF4-, BGLF4-KD-, stathmin-4E, stathmin-Flag, or stathmin-4A-expresion cells) were compared with that in vector control cells by using the 2 × 2 Contingency Table of chi-squared test. *, significant difference to vector control (p < 0.05).

FIGURE 6.

UL13 homologues induce phosphorylation of stathmin in vivo. HeLa cells were transiently transfected with vector and Flag-tagged UL13 (HSV-1), UL97 (HCMV), mORF36 (MHV68), or ORF36 (KSHV)-expressing plasmids. A BGLF4 expression plasmid was transfected as the positive control. Cell lysates were harvested 24 h post-transfection and subjected to immunoblotting analysis. The membrane was probed with specific antibodies against BGLF4, Flag, phosphoserine 16 stathmin, and GAPDH. The blot was then stripped and re-probed with anti-C-terminal stathmin Ab. The band densities of nonphosphorylated or total phosphorylated stathmin of each transfection relative to the vector control were quantified and normalized to the intensity of its corresponding internal control by using ImageQuant software. Relative densities are indicated below the corresponding panels, and relative densities of phosphorylated stathmin from three independent experiments are statistically analyzed and plotted. *, significant difference to vector control (p < 0.05); **, significant difference to vector control (p < 0.01).