Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase.
Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rho-kinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasin-dependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.
Amano
Mutsuki
M
Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Aichi, Japan.
Kaneko
Takako
T
Maeda
Akio
A
Nakayama
Masanori
M
Ito
Masaaki
M
Yamauchi
Takashi
T
Goto
Hideyuki
H
Fukata
Yuko
Y
Oshiro
Noriko
N
Shinohara
Azusa
A
Iwamatsu
Akihiro
A
Kaibuchi
Kozo
K
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D016466:CHO Cells; D006224:Cricetinae; D015870:Gene Expression; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D054460:rho-Associated Kinases; D016875:tau Proteins / Q000235:genetics / Q000378:metabolism*
D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D021122:Protein Subunits; D016875:tau Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D043263:Myosin-Light-Chain Phosphatase