The N-terminal LIM domain negatively regulates the kinase activity of LIM-kinase 1.
LIM-kinase 1 (LIMK1, where LIM is an acronym of the three gene products Lin-11, Isl-1 and Mec-3) is a serine/threonine kinase that phosphorylates cofilin and regulates actin cytoskeletal reorganization. LIMK1 contains two LIM domains and a PDZ (an acronym of the three proteins PSD-95, Dlg and ZO-1) domain in the N-terminal half and a kinase domain in the C-terminal half. In this study we examined the role of the extra-catalytic region in the regulation of kinase activity of LIMK1. Limited proteolysis of LIMK1 resulted in the production of the 35-40-kDa kinase core fragments with 3.5-5. 5-fold increased kinase activity. The LIMK1 mutants with deleted LIM domains (DeltaLIM) or conserved cysteines in the two LIM domains replaced with glycines (dmLIMK1) had 3-7-fold higher kinase activities in vitro, compared with the wild-type LIMK1. The C-terminal kinase fragment of LIMK1 bound to the LIM domain but not to the PDZ domain. Furthermore, the LIM fragment dose-dependently inhibited the kinase catalytic activity of the kinase core fragment of LIMK1. Taken together, these results suggest that the N-terminal LIM domain negatively regulates the kinase activity of LIMK1 by direct interaction with the C-terminal kinase domain. In addition, expression of the DeltaLIM mutant in cultured cells induced punctate accumulation of actin filaments, an event distinct from the pattern of actin organization induced by expression of the wild-type LIMK1, suggesting that the LIM domain plays a role in the function of LIMK1 in vivo.
Nagata
K
K
Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Ohashi
K
K
Yang
N
N
Mizuno
K
K
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D003599:Cytoskeleton / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D054461:Lim Kinases; D008969:Molecular Sequence Data; D017354:Point Mutation; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013379:Substrate Specificity
D000199:Actins; D004268:DNA-Binding Proteins; D011494:Protein Kinases; C515892:LIMK1 protein, human; D054461:Lim Kinases; D017346:Protein Serine-Threonine Kinases