|Amano, T., Tanabe, K., Eto,
T., Narumiya, S. & Mizuno, K. (2001) LIM-kinase 2 induces formation
of stress fibres, focal adhesions and membrane blebs, dependent on its
activation by Rho-associated kinase-catalysed phosphorylation at
threonine-505. Biochem. J. 354, 149-159.
|LIM-kinase 1 and 2 (LIMK1 and LIMK2)
phosphorylate cofilin and induce actin cytoskeletal reorganization.
LIMK1 is activated by Rho-associated, coiled-coil-forming protein kinase
(ROCK) and p21-activated kinase 1 (PAK1), but activation mechanisms and
cellular functions of LIMK2 have remained to be determined. We report
here that LIMK1 and LIMK2 phosphorylate both cofilin and
actin-depolymerizing factor (ADF) specifically at Ser-3 and exhibit
partially distinct substrate specificity when tested using site-directed
cofilin mutants as substrates. We also show that LIMK2 is activated by
ROCK by phosphorylation at Thr-505 within the activation loop. Wild-type
LIMK2, but not its mutant (T505V) with replacement of Thr-505 by Val,
was activated by ROCK in vitro and in vivo. LIMK2 mutants with
replacement of Thr-505 by one or two Glu residues (T505E or T505EE)
increased the kinase activity about 3.6-fold but were not further
activated by ROCK. When expressed in HeLa cells, wild-type LIMK2, but
not the T505V mutant, induced the formation of stress fibres, focal
adhesions and membrane blebs. Furthermore, inhibitors of Rho and ROCK
significantly suppressed LIMK2-induced stress fibres and membrane blebs.
These results suggest that LIMK2 functions downstream of the Rho-ROCK
signalling pathway and plays a role in reorganization of actin filaments
and membrane structures, by phosphorylating cofilin/ADF proteins.