The phosphorylation of p25/TPPP by LIM kinase 1 inhibits its ability to assemble microtubules.
LIM kinase 1 (LIMK1) is a key regulator of actin dynamics as it phosphorylates and inactivates cofilin, an actin-depolymerizing factor. LIMK1 activity is also required for microtubule disassembly in endothelial cells. A search for LIMK1-interacting proteins identified p25alpha, a phosphoprotein that promotes tubulin polymerization. We found that p25 is phosphorylated by LIMK1 on serine residues in vitro and in cells. Immunoblotting analysis revealed that p25 is not a brain specific protein as previously reported, but is expressed in all mouse tissues. Immunofluorescence analysis demonstrated that endogenous p25 is co-localized with microtubules and is also found in the nucleus. Down-regulation of p25 by siRNA decreased microtubule levels while its overexpression in stable NIH-3T3 cell lines increased cell size and levels of stable tubulin. Bacterially expressed unphosphorylated p25 promotes microtubule assembly in vitro; however, when phosphorylated in cells, p25 lost its ability to assemble microtubule. Our results represent a surprising connection between the tubulin and the actin cytoskeleton mediated by LIMK1. We propose that the LIMK1 phosphorylation of p25 blocks p25 activity, thus promoting microtubule disassembly.
Acevedo
Karla
K
St. Vincent's Institute of Medical Research, 9 Princes St. Fitzroy, Victoria 3065, Australia.
Li
Rong
R
Soo
Priscilla
P
Suryadinata
Randy
R
Sarcevic
Boris
B
Valova
Valentina A
VA
Graham
Mark E
ME
Robinson
Phillip J
PJ
Bernard
Ora
O
D000818:Animals; D048429:Cell Size; D015536:Down-Regulation; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D054461:Lim Kinases / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009928:Organ Specificity; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012756:Sheep; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D014404:Tubulin / Q000378:metabolism
D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C118136:TPPP protein, human; D014404:Tubulin; D010768:Phosphoserine; D054461:Lim Kinases