In the present study, we found that 14-3-3β directly binds to MYPT1. It has been shown that 14-3-3 can interact with various proteins and influence the localization of target molecules and/or regulate the enzymatic activity (Aitken, 1995
; Fu et al., 2000
; Muslin and Xing, 2000
; van Hemert et al., 2001
; Tzivion and Avruch, 2002
; Aitken, 2006
). We found that 14-3-3β attenuates the phosphatase activity of MLCP, but not the catalytic subunit. Because the binding of 14-3-3β to MYPT1 does not interfere with the intersubunit binding of the MLCP holoenzyme, the 14-3-3β–induced decrease in the phosphatase activity would be due to the change in the conformation at the binding sites between MYPT1 and the catalytic subunit. It is known that MYPT1 significantly increases the myosin phosphatase activity of PP1δ and that the Rho-kinase induced phosphorylation attenuates the elevated phosphatase activity without dissociation of MYPT1 from the holoenzyme (Feng et al., 1999
). These results suggest that the inhibition mechanism of MYPT1 by 14-3-3β is similar to the inhibition by MYPT1 phosphorylation in which the inhibition is achieved by attenuating the activator function of MYPT1. It is plausible that the binding of 14-3-3β to MYPT1 changes the binding interface between MYPT1 and PP1δ thus abolishing the critical interaction between MYPT1 and PP1δ that affects the catalytic activity.
Quite interestingly, we found that 14-3-3β binding to MYPT1 induces the dissociation of MYPT1 from myosin. Consistently, the overexpression of 14-3-3β in cells markedly diminished the localization of MYPT1 at the stress fiber. Because MYPT1 associates with myosin II in the stress fiber, the result can be explained by the finding that the binding of 14-3-3β to MYPT1 induces dissociation of MYPT1 from myosin II in stress fiber. Because 14-3-3β does not break the MLCP subunit structure, it is anticipated that 14-3-3β dissociates the MLCP holoenzyme from myosin by breaking the interaction between MYPT1 and myosin. The dissociation of MLCP from myosin is expected to result in decrease in the dephosphorylation rate of myosin, thus increasing the myosin phosphorylation level. It should be noted that 14-3-3 inhibited the phosphatase activity of MLCP even though the isolated MLC was used as a substrate, suggesting that the inhibition of the phosphatase activity in vitro is not directly due to the dissociation of MLCP from myosin. Therefore, it is anticipated that 14-3-3 inhibits MLCP activity by dual mechanism in cells, i.e., the decrease in the enzymatic activity and the segregation of the substrate (myosin) from the enzyme (MLCP). Supporting this view, we actually found that the expression of 14-3-3β increased the MLC phosphorylation level in cells (), and this is due to the down-regulation of myosin phosphatase, but not the activation of myosin kinase activity.
It has been puzzling that although isolated MLCP strongly binds to myosin, the majority of MLCP in cell is localized throughout cytosol, and only a part of the MLCP colocalized with the structure where myosin II is present, such as stress fiber (Murata et al., 1997
). The present results suggest that 14-3-3β is at least, in part, responsible for the cytosolic localization of MLCP. The question is how 14-3-3β controls MLCP localization at the myosin II–containing structure in vivo. To address this question, we examined the effect of the protein phosphatase treatment of MYPT1 on the binding to 14-3-3β. The result suggested that the phosphorylation of MYPT1 is critical for the binding of MYPT1-14-3-3β.
Further analysis revealed that the motif RSXSXP present in the MYPT1 sequence is responsible for the interaction between 14-3-3β and MLCP, because 1) the mutation of S472 of MYPT1 inhibited the growth of the 14-3-3β–transfected yeast on the nutrient selection plates in yeast two-hybrid experiments and 2) the S472A and S472D mutant of MYPT1 failed to coimmunoprecipitate with 14-3-3β in contrast to the WT MYPT1. This view is supported by the finding that the localization of the S472A mutant of MYPT1 at the stress fiber in COS7 cells was not influenced by 14-3-3β expression, whereas the stress fiber localization of WT MYPT1 was diminished. It has been reported that 14-3-3 protein bind to the phosphorylated proteins and the RSXSXP motif is critical for the binding to partner proteins such as Raf (Muslin et al., 1996
) and CDC25 (Zha et al., 1996
; Mils et al., 2000
). The present result agrees with the notion found in other 14-3-3 binding proteins.
Because the mutation of S472 interferes with the interaction between 14-3-3β to MYPT1, the phosphorylation site critical for the binding should be S472. To clarify this issue, we produced the antibody that specifically recognize the phosphorylated Ser472 of MYPT1. The antibody recognized MYPT1 phosphorylated by Rho-kinase, but not MYPT1 dephosphorylated by PP1δ. Furthermore, the mutation of Ser472 completely abolished the interaction of these antibodies with MYPT1. These results indicate that the produced antibodies are specific to the phosphorylated Ser472 of MYPT1. Interestingly, the isolated MYPT1 was recognized with anti-phospho-Ser472 antibody, indicating that MYPT1 expressed in Sf9 cells is partially phosphorylated at Ser472. Therefore, it is thought that the binding of 14-3-3 to the expressed MYPT1 is because of the presence of phosphorylated MYPT1 at Ser472.
It is interesting that S472D MYPT1 also failed to interact with 14-3-3β, indicating that the introduction of the negative charge at the position of Ser472 does not mimic the phosphorylation effect. The introduction of the acidic residues has been used to determine the phosphorylation sites of proteins assuming that the negative charges of acidic amino acid resemble the phosphate moiety, thus mimicking the phosphorylation effect. However, the 3D position of the phosphate moiety is different from that of the side chains of the acidic residues, and it is anticipated that the acidic residues do not always mimic the phosphorylation effect. In fact, the replacement of Ser19 of MLC by Asp does not mimic the phosphorylation effect on myosin motor function, although it mimics the phosphorylation effect on filament formation of myosin (Ikebe et al., 1994a
; Kamisoyama et al., 1994
). It was also shown that mutation of S175 of calponin to Asp shows a dephosphorylated phenotype rather than the phosphorylated phenotype (Tang et al., 1996
). We think that the introduction of a phosphate moiety at a proper 3D configuration is essential to induce the binding of MYPT1-14-3-3β.
Using the phosphorylation site specific antibody against Ser472, we found that Rho-kinase phosphorylates Ser472 of MYPT1 in cells, and this is consistent with the previous report that Rho-kinase can phosphorylate this site in vitro (Kawano et al., 1999
). Furthermore, we found that Ser472 MYPT1 phosphorylation is increased by EGF stimulation, and the Ser472 phosphorylation is inhibited by the Rho-kinase inhibitor. Therefore, it is plausible that the activation of the RhoA pathway induces the phosphorylation of MYPT1 at Ser472, which enhances the interaction between 14-3-3β and MYPT1. Surprisingly, the elimination of endogenous Rho-kinase I and II by Rho kinase–specific siRNA did not affect EGF-induced Thr799 MYPT1 phosphorylation, whereas the siRNA-induced gene silencing diminished both the EGF-induced increase in the Ser472 MYPT1 phosphorylation and MLC phosphorylation. Consistently, 14-3-3β inhibited T641A/T799A MLCP activity to the same potency as the inhibition of WT MLCP activity. These results indicate that Ser472 but not Thr641/Thr799 MYPT1 phosphorylation is predominantly regulated by Rho-kinase in the EGF signaling, thus regulating MLCP activity and MLC phosphorylation. Further studies are required to clarify the pathways responsible for the regulation of MYPT1 phosphorylation at Thr641/Thr799 sites.
On the basis of the present study, we propose the following scenario for the regulatory function of 14-3-3/MYPT1. The agonist stimulates RhoA/Rho-kinase pathway, which phosphorylates Ser472 MYPT1. The phosphorylation enhances the interaction between 14-3-3β and MYPT1, which results in the decrease in the myosin dephosphorylation rate due to the inhibition of MLCP activity and the dissociation of MLCP from myosin, thus increasing in myosin phosphorylation (). It has been known that the activation of RhoA pathway increases myosin phosphorylation due to the inhibition of myosin dephosphorylation activity (Kimura et al., 1996
). The present study reveals the novel mechanism of RhoA-dependent MLCP regulatory mechanism and suggests that the binding of 14-3-3 to MYPT1 is, in part, responsible for the down-regulation of MLCP, thus enhancing myosin II–based motor activity in mammalian cells.
Figure 10. A model for the regulation of MLCP by 14-3-3. MLCP is associated with myosin II via MYPT1/myosin II heavy-chain interaction. Once MYPT1 is phosphorylated at Ser472, 14-3-3 binds to MYPT1, dissociating MLCP from myosin II heavy chain and decreasing the (more ...)