Phosphorylation of Microtubule-associated Protein SB401 from Solanum berthaultii Regulates Its Effect on Microtubules

Casein kinase II phosphorylates SB401

Analysis of the SB401 protein sequence predicts that there are three sites for CKII phosphorylation in the protein domain that contains the VEEKKEE repeats, which are important for the binding of SB401 to MTs (http://www.predictprotein.org/) (Figure 1). This suggests that CKII may be involved in the regulation of SB401 activity.

First, we used a commercial CKII to test whether CKII could phosphorylate SB401. When SB401 was incubated with CKII and [γ-³²P] ATP, an apparent signal was observed (Figure 2A, B, lane 2). However, no signal was detected when SB401 was incubated with [γ-³²P] ATP in the absence of CKII (Figure 2A, B, lane 1), or with CKII (Figure 2A, lane 3). Bovine serum albumin (BSA), which was used as a negative control, was not phosphorylated by CKII (Figure 2A, B, lane 4). This result demonstrates that CKII is capable of phosphorylating SB401.

Phosphorylation of SB401 downregulates its activity in bundling microtubules and enhancing the polymerization of tubulin

Further experiments were carried out to investigate the effect of phosphorylation of SB401 on its involvement in the bundling of MTs.

The activity of SB401 in influencing the bundling of MTs was tested. The MTs were preformed in the presence of 10 μmol/L taxol. The MTs that had been stabilized by taxol were incubated with or without SB401, or P-SB401 (phosphorylated SB401), respectively, before observation using confocal microscopy. The results showed that taxol-stabilized MTs without the addition of SB401 appeared in a pattern of single filaments (Figure 3A). When 2 μmol/L of SB401 was added, MT bundles formed (Figure 3B). However, no MT bundles were observed when 2 μmol/L of P-SB401 was added to the preformed MTs (Figure 3C). This observation indicates that the phosphorylation of SB401 downregulates its activity with respect to the bundling of MTs.

The activity of SB401 in the enhancement of tubulin polymerization was also investigated. The tubulins were polymerized in the absence or presence of varying concentrations of SB401. The polymerization was monitored by turbidimetric assay. The results showed that the polymerization of tubulin was enhanced by the addition of SB401 in a concentration-dependent manner (Figure 4). In contrast, when P-SB401 (phosphorylated SB401) was used in the experiments, the polymerization of tubulin was downregulated (Figure 4).

In conclusion, our tests demonstrated that the effects of SB401 on MTs were altered when SB401 was phosphorylated by CKII.

Phosphorylation of SB401 has no effect on its activity in bundling F-actin

Our previous report provided evidence that SB401 also binds to and bundles F-actin (Huang et al. 2007). Therefore, the effect of SB401 on the bundling of F-actin was also tested. F-actin was preformed in the presence of 0.5 μmol/L phalloidin. Phalloidin-stabilized F-actin appeared in a pattern of single filaments (Figure 5A). When 2 μmol/L of SB401 was added, bundles of F-actin were formed (Figure 5B). This effect was also observed when 2 μmol/L of P-SB401 was added (Figure 5C). Therefore, the phosphorylation of SB401 has no effect on its activity in the bundling of F-actin.

A CKII-like kinase exists in potato pollen extract

The experimental results described above indicate that phosphorylation of SB401 by CKII downregulated its involvement in the bundling of MTs and enhancement of the polymerization of tubulin. We also wished to investigate whether any CKII-like kinase in cells exerts such an effect. Given that SB401 is expressed in potato pollen and is enriched in extracts of mature pollen grains and in pollen that is germinated in vitro (Liu et al. 1997), we used extracts from potato pollen grains and pollen that was germinated in vitro to test whether any CKII-like kinase was present. In order to identify CKII-like kinase from the potato pollen extract (PE), an anti-CKII alpha antibody was used in Western blotting experiments. A major protein band with a molecular mass of 55 kDa was recognized specifically in the PE by the antibody (Figure 6A, B, lane 2). No signal was detected from BSA, which was used as a control (Figure 6A, B, lane 1). The molecular mass of the protein identified herein is different from those reported for Arabidopsis (45 kDa, Espunya and Martinez 1997), broccoli (48 kDa, Klimczak and Cashmore 1994), and pea (41 kDa, Zhang et al. 1993). This finding suggests that different isoforms of CKII-like kinases exist in plants.

In addition, a phosphorylation assay was carried out to test whether there is any kinase activity that influences the SB401 protein in PE. When SB401 was incubated with PE and [γ-³²P] GTP, an apparent signal was detected (Figure 6C, D, lane 2). However, no signal was detected when SB401 was incubated with [γ-³²P] GTP in the absence of PE (Figure 6C, D, lane 1). A signal was also detected in the PE, which suggests that the endogenous SB401 was phosphorylated (Figure 6C, D, lane 3). These observations suggest that CKII-like kinase is present in potato pollen extract.

Potato pollen extract downregulates SB401 involvement in bundling of microtubules

To further elucidate whether phosphorylation of SB401 by pollen extract had an effect on the bundling of MTs similar to that described above, we pre-incubated SB401 with PE and ATP to allow the SB401 protein to be phosphorylated and then tested its effect on the bundling of MTs.

Preformed taxol-stabilized MTs appeared in a pattern of single filaments (Figure 7A). The addition of 3 μmol/L of SB401 caused bundling of the MTs (Figure 7B). The addition of PE (0.2 mg/mL), without or with ATP (50 μmol/L), had no effect on MT bundling (Figure 7C, D). Therefore, PE alone was not involved in the bundling of MTs. When 3 μmol/L of SB401 was added together with PE (0.2 mg/mL), the MTs formed bundles (Figure 7E). The addition of ATP had no effect on this formation of bundles by SB401 (Figure 7F). However, no MT bundles were observed in the presence of SB401, PE, and ATP (Figure 7G), which suggests that PE phosphorylated the SB401 and consequently downregulated its effect on bundling of MTs. To confirm this, the CKII-like kinase was removed from PE by immunodepletion. In the presence of SB401, AbB (PE in which CKII-like-kinase was immunodepleted) and ATP, the MTs formed bundles (Figure 7H). Pretreatment of the PE with protein A-sepharose beads without antibody (ckB) had no effect on MT bundling (Figure 7I). Therefore, the CKII-like kinase that was present in the PE was responsible for the phosphorylation of SB401 and therefore downregulated its involvement in the formation of MT bundles.

Phosphorylation of SB401 switches its functional activity between MTs and F-actin

Phosphorylation plays an important role in the regulation of the activities of MAPs. Phosphorylation of XMAP215 reduces its activity in the regulation of MT dynamics (Vasquez et al. 1999). Phosphorylation of Tau at S²⁶² inhibits its activity in bundling of MTs, whereas phosphorylation at S²¹⁴ inhibits its nucleation activity in the polymerization of tubulin (Yoshida et al. 2004). A number of studies have demonstrated that phosphorylation by various kinases of proteins that bind and stabilize MTs is an important mechanism for the regulation of mitosis (Yarm 2002; Rong et al. 2007). In plant cells, it has also been reported that Arabidopsis AtMAP65-1 and tobacco NtMAP65-1 are phosphorylated during the cell cycle (Sasabe et al. 2006; Smertenko et al. 2006).

We have reported previously that SB401 from S. berthaultii is able to bind to both MTs and F-actin, which suggests that SB401 may function as a link between actin and the MT cytoskeleton (Huang et al. 2007). However, SB401 binds preferentially to MTs. There must be some mechanism to regulate the switch of the function of SB401 between MTs and F-actin. In the study reported herein, we demonstrated that phosphorylation may play a role in such regulation. The phosphorylation of SB401 downregulates its involvement in the bundling of MTs and the enhancement of the polymerization of tubulin. In contrast, such phosphorylation has no effect on the involvement of SB401 in the bundling of F-actin. Therefore, phosphorylated SB401 may function mainly in association with F-actin, whereas dephosphorylated SB401 may act mainly on MTs.

Regulation by phosphorylation is also found in other proteins that bind to both MTs and F-actin. For instance, MAP1B, which also contains VEEKKEE repeats and binds to both MTs and F-actin, has at least one Proline Directed Protein Kinase (PDPK) and two CKII phosphorylation sites. Phosphorylation of purified MAP1B results in the loss of its ability to bind to F-actin, whereas dephosphorylation restores this ability (Pedrotti et al. 1994; Gong et al. 2000; Riedere 2007). It appears that phosphorylation of MAP1B and SB401 by CKII has different effects on F-actin.

Phosphorylation of SB401 regulates its conformation

Our experimental results demonstrated that the phosphorylation of SB401 downregulates its involvement in the formation of MT bundles and in enhancement of the polymerization of tubulin. How is this regulation achieved?

Analysis of the structure predicted by the domain of SB401 protein (http://www.predictprotein.org/), which contains six VEEKKEE repeats and three CKII phosphorylation sites, suggests that this protein sequence has structurally ambivalent sequence fragments. These are able to adopt either helical or sheet conformations, and a limited number of substitutions results in the conversion of a helical protein to a protein of predominantly sheet formation (Kuznetsov and Rackovsky 2003). The structurally ambivalent fragments in the SB401 protein may be the key to the regulation of bundling of MTs. For example, the phosphorylation of Ser⁶³ in Op18/stathmin disrupts the central helix and suppresses its binding to tubulin (Steinmetz et al. 2001). Phosphorylation modifies both the negative charge and the local conformation near the phosphorylation sites in Tau protein (Du et al. 2005). These structural changes suggest that phosphorylation may act as a conformational switch in the binding domain of MAPs. Phosphorylation is one strategy by means of which control of biological activity can be enforced.

Further investigations are required to identify the CKII-like kinase in potato pollen, to elucidate how the phosphorylation of SB401 functions within the cell, and to investigate the physiological role of the phosphorylation of SB401. Nevertheless, the current study has provided evidence to show that CKII-like kinase activity exists in potato pollen, and that phosphorylation of SB401 affects its function with regard to MTs. These results will provide the basis for our future studies.

In vitro pollen germination and preparation of pollen extracts

Pollen grains were collected from mature flowers of Solanum berthaultii and stored at −80 °C. The pollen grains were spread in liquid germination medium (20 mmol/L Mes-potassium hydroxide (KOH) pH 6.0, 0.01% (w/v) KNO₃, 0.02% MgSO₄-7H₂O, 0.01% H₃BO₄, 0.07% Ca(NO₃)₂-4H₂O, 2% sucrose, 15% PEG6000) and germinated as reported previously (Liu et al. 1997).

To prepare the protein extract from the germinated pollen, potato pollen grains (∼0.2 g) were collected by centrifugation at 12 000g for 10 min and homogenized with a mortar and pestle under liquid nitrogen. The mixture was resuspended with 2–3 mL of extraction buffer. After centrifugation at 50 000g for 40 min, the supernatants were transferred into clean tubes and kept in an iced-water bath. The protein concentration of the pollen extract was determined by a Bio-Rad Laboratories (Hercules, CA, USA) protein assay with BSA as the standard. Two extraction buffers were used for the preparation. The extraction buffer for the kinase assay contained 100 mmol/L Hepes (pH 7.5), 5 mmol/L ethylene diamine tetraacetic acid (EDTA, pH 8.0), 5 mmol/L ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, pH 7.0), 10 mmol/L NaF, 50 mmol/L glycerophosphate (sodium salt), 5% (v/v) glycerol, 10 mmol/L Na₃VO₄, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mmol/L phenylmethanesulfonyl fluoride (PMSF), and 10 mmol/L dithiothreitol (DTT) (Zhang and Liu 2001).

The extraction buffer for the MT assay contained 100 mmol/L Pipes, 1 mmol/L EGTA (pH 8.0), 1 mmol/L MgCl₂, 10 μg/mL aprotinin, and 10 μg/mL leupeptin, at pH 6.9, adjusted with KOH.

Antibodies and protein gel blots

The protein samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to nitrocellulose membranes. Blots were probed with a polyclonal antibody to CKII alpha subunit (ab13410, Abcam, Cambridge, UK) at a dilution of 1:500 with TBST (50 mmol/L Tris, 150 mmol/L NaCl, and 0.05% Tween 20, pH 7.5). The secondary antibody was horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA), applied at a dilution of 1:10 000.

Phosphorylation assays

Recombinant SB401 protein was prepared as reported previously (Huang et al. 2007). Phosphorylation assays were prepared using commercial CKII kits (#P6010S, New England Labs, Beverly, MA, USA). Briefly, 3 μg SB401 was phosphorylated in a reaction system (30 μL) that contained CKII reaction buffer (20 mmol/L Tris-HCl, pH 7.5, 50 mmol/L KCl, 10 mmol/L MgCl₂, 3 μCi [γ-³²P] ATP, and 50 μmol/L ATP) in the presence of 5–10 U commercial CKII at 25 °C for 30 min. The protein samples were separated by SDS-PAGE. The gels were stained using Coomassie brilliant blue, dried, and subjected to autoradiography. BSA (3 μg) was used as the negative control.

To phosphorylate the SB401 protein, 10 μg of pollen protein extract in PE buffer was mixed with 3 μg of SB401 in the reaction system (30 μL) and incubated with 3 μCi [γ-³²P] of GTP at 25 °C for 20 min; 50 μmol/L of GTP was then added to the solution and incubated for an additional 60 min. The protein samples were then separated by SDS-PAGE. The gels were stained using Coomassie brilliant blue, dried, and subjected to autoradiography.

Microtubule polymerization assay

Porcine brain tubulin was prepared as reported previously (Huang et al. 2007). To monitor the time course of tubulin polymerization, various concentrations (0.0, 0.2, 0.4, or 0.8 μmol/L) of SB401 protein or phosphorylated SB401 protein (0.2 or 0.4 μmol/L) were added to 50 μmol/L of tubulin in PEM (100 mmol/L PIPES, 1 mmol/L EGTA, 1 mmol/L MgCl₂) buffer containing 1 mmol/L of GTP. Microtubule polymerization was monitored spectrophotometrically at OD₃₅₀ in a 0.4 cm-wide quartz cuvette at 35 °C for 30 min using a DU-640 spectrophotometer (Beckman Coulter, Fullerton, CA, USA). Each experiment was repeated at least two times.

Microtubule and F-actin bundling assays

Fluorescence labeling experiments were carried out in vitro to investigate the bundling of SB401 to MTs and F-actin. NHS-rhodamine (5(and-6)carboxytetra-methyl-rhodamine succinimidyl ester) labeled tubulin was prepared as reported previously (Huang et al. 2007). Purified recombinant SB401 (2 μmol/L) or P-SB401 (2 μmol/L) was incubated with taxol-stabilized, rhodamine-labeled MTs (0.5 μmol/L) in 50 μL PEM buffer at room temperature in a tube by gently pipetting with a cut tip at room temperature for 5 min. One microliter of the mixture was gently pipetted on to the surface of a glass slide, gently covered with a cover slip, and observed using a confocal microscope (Meta 510, Zeiss, Jena, Germany) with a Zeiss 40× magnification oil objective (NA 1.3).

Rabbit muscle actin was prepared as previously reported (Huang et al. 2007). F-actin (0.5 μmol/L) polymerized with 100 nmol/L of Alexa-488 phalloidin was incubated with 2 μmol/L of SB401 protein or 2 μmol/L of P-SB401 protein at room temperature for 5 min. Aliquots (1 μL) of the samples were placed on to a slide and observed using the confocal microscope.

Immunodepletion

The immunodepletion experiment was carried out as described by Lee et al. (1998). Ten milligrams of protein A sepharose CL-4B beads (GE Healthcare, Uppsala, Sweden) were swollen in 1 mL fresh distilled water. After the beads had settled by gravity, the supernatant was drawn off and the medium was resuspended with 1 mL of fresh distilled water. This procedure was repeated six times. The beads were then transferred into PEM buffer and washed with 1 mL PEM buffer five times. The settled medium was used in the immunodepletion experiments. Approximately 200 μg of pollen extract and 5.5 μg of commercial CKII antibody were mixed and incubated overnight at 4 °C. Subsequently, 5 μL of pre-washed protein A-sepharose bead slurry was added to the mixture and incubated for 5 h. Immunodepleted supernatants (AbB) were obtained by centrifugation at 12 000g for 90 s. Those treated with protein A-sepharose bead without antibody were used as a negative control, designated ckB.

(Handling editor: Chun-Ming Liu)