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, 14 (5), 1745-56

Citron Kinase, a Rho-dependent Kinase, Induces Di-Phosphorylation of Regulatory Light Chain of Myosin II

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Citron Kinase, a Rho-dependent Kinase, Induces Di-Phosphorylation of Regulatory Light Chain of Myosin II

Shigeko Yamashiro et al. Mol Biol Cell.

Abstract

Citron kinase is a Rho-effector protein kinase that is related to Rho-associated kinases of ROCK/ROK/Rho-kinase family. Both ROCK and citron kinase are suggested to play a role in cytokinesis. However, no substrates are known for citron kinase. We found that citron kinase phosphorylated regulatory light chain (MLC) of myosin II at both Ser-19 and Thr-18 in vitro. Unlike ROCK, however, citron kinase did not phosphorylate the myosin binding subunit of myosin phosphatase, indicating that it does not inhibit myosin phosphatase. We found that the expression of the kinase domain of citron kinase resulted in an increase in MLC di-phosphorylation. Furthermore, the kinase domain was able to increase di-phosphorylation and restore stress fiber assembly even when ROCK was inhibited with a specific inhibitor, Y-27632. The expression of full-length citron kinase also increased di-phosphorylation during cytokinesis. These observations suggest that citron kinase phosphorylates MLC to generate di-phosphorylated MLC in vivo. Although both mono- and di-phosphorylated MLC were found in cleavage furrows, di-phosphorylated MLC showed more constrained localization than did mono-phosphorylated MLC. Because citron kinase is localized in cleavage furrows, citron kinase may be involved in regulating di-phosphorylation of MLC during cytokinesis.

Figures

Figure 1.
Figure 1.
Schematic diagram of citron kinase, ROK and ROCK and their truncation mutants. Modified from Figure 1 of Madaule et al. (1998) with permission.
Figure 2.
Figure 2.
Phosphorylation of RMLC by citron-kinase in vitro. (A) Phosphorylation of MLC by citron-kinase. Citron kinase Δ1 mutant was expressed in COS7 cells, immunoprecipitated with an antimyc antibody, and assayed for phosphorylation using bovine lung myosin II (lanes 2 and 3) or isolated MLC (lanes 4–6) as a substrate. Lanes 1 and 2, Coomassie blue staining. Lane 1, molecular mass markers (numbers in kilodaltons); lane 2, lung myosin plus immunoprecipitated kinase; lane 3, autoradiograph of lane 2. Lanes 4–6, autoradiograph; lane 4, immunoprecipitates from mock-transfected cells; lane 5, immunoprecipitates of citron kinase; lane 6, immunoprecipitates of kinase-defective citron kinase. HC, heavy chain of myosin II; MLC, myosin light chain. (B) One-dimensional phosphopeptide map. Citron kinase phosphorylated both Ser19 and Thr18. Note that citron kinase produced di-phosphorylated MLC to a higher extent than did MLCK. Protein kinase C phosphorylated Ser1/2 and Thr9.
Figure 3.
Figure 3.
Citron kinase phosphorylates mono-phosphorylated MLC as well as it does un-phosphorylated MLC. MLC kinase reactions were performed using either unphosphorylated myosin (0P) or mono-phosphorylated myosin (1P) as a substrate. Myc-tagged, full-length citron kinase, Δ2 citron kinase, or Δ3 ROCK was expressed in COS7 cells, immunoprecipitated by a myc antibody, and used for kinase reactions. Purified MLCK was also used. Ratios of phosphate incorporation between mono-phosphorylated MLC and unphosphorylated MLC are shown. Kinase levels are shown in the bottom panel (Coomassie blue staining). The results are representative of three independent experiments.
Figure 4.
Figure 4.
Citron kinase does not significantly phosphorylate MBS. The kinase domain mutants (Δ2) of citron kinase and ROCK (Δ3) were expressed in COS7 cells, immunoprecipitated, and assayed for their kinase activities using MBS (a) or MLC (b) as a substrate. Both citron kinase (lane 2) and ROCK (lane 3) phosphorylated MLC to a similar extent (b). However, only ROCK phosphorylated MBS (lane 3 of a). Lane 1 is control without kinase. The levels of kinase used are shown in c, which were detected by immunoblot using the antimyc antibody. CBB, Coomassie brilliant blue. The results are representative of three independent experiments.
Figure 5.
Figure 5.
Phosphorylation of MLC by Baculovirus-expressed full-length citron kinase. Full-length citron kinase (5 ng/μl) was incubated at 30°C for 5 min with varying concentrations (0.25–15 μM) of MLC. Pi incorporation is in pmol.
Figure 6.
Figure 6.
Di-phosphorylation of MLC in vivo by citron kinase. PTK cells were first transfected with Δ2 citron kinase and then immunolabeled with the specific antibodies against myc (A, D, G, and J), against mono-phosphorylated MLC (B and H), and against di-phosphorylated MLC (E, I, and K). F-actin was also visualized by labeling with fluorescent phalloidin (C, F, and L). Asterisks indicate cells expressing myc-tagged mutant citron kinase. Cells in J–L were treated with a specific inhibitor of ROCK, Y-27632, for 30 min before immunofluorescence staining. The expression of Δ2 citron kinase increased di-phosphorylation of MLC (asterisks in E, I, and K) but decreased mono-phosphorylation (asterisk in B and H). Bar, 10 μm.
Figure 7.
Figure 7.
Increases in mono- and di-phosphorylation by the expression of ROCK. PTK cells were transfected with Δ3 mutant of ROCK (equivalent to Δ2 mutant of citron kinase) and then stained with antimyc (A and D), anti–mono-phosphorylated MLC (B), and anti–di-phosphorylated MLC (E) antibodies. F-actin structure was visualized by labeling with fluorescent phalloidin (C and F). Cells expressing myc-tagged mutant ROCK are indicated by asterisks. Note that ROCK increases both mono- and di-phosphorylation of MLC. Stellar stress fibers were frequently formed. Bar, 10 μm.
Figure 8.
Figure 8.
Increase in di-phosphorylation and decrease in mono-phosphorylation of MLC by expression of citron kinase. BHK cells were first transfected with Δ2 mutant of citron kinase or Δ3 mutant of ROCK, then total cell lysates were immunoblotted with antibodies against mono-phosphorylated MLC (middle panel of a) or against di-phosphorylated MLC (middle panel of b). Lane 1, mock-transfection; lane 2, transfection with Δ 2 citron kinase mutant; lane 3, transfection with Δ3 ROCK mutant. The same lysates were also immunoblotted with antimyc antibody to show levels of expression (top panels). Coomassie blue staining of histone H3 of total cell lysates are shown in the lower panels to indicate the loading.
Figure 9.
Figure 9.
Di-phosphorylation of MLC during cytokinesis by the expression of full-length citron kinase. CHO cells were transfected with GFP-full-length citron kinase (A–C) or GFP alone (D–F) and synchronized for cell division. A and D, GFP. Cleaving cells were labeled with the anti–di-phosphorylated MLC antibody (B and E) and with DAPI (C and F).
Figure 10.
Figure 10.
Localization of mono- and di-phosphorylated MLC during cell division. NRK cells at different stages of cell division were stained with the antibodies against mono-phosphorylated MLC (A, D, and G), di-phosphorylated MLC (B, E, and H), and DAPI (C, F, and I). A–C, early anaphase; D–F, telophase; G–I, late telophase. Note that di-phosphorylated MLC in cleavage furrows is more constrained than is mono-phosphorylated MLC. Bar, 10 μm.

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