Human CLP36, a PDZ-domain and LIM-domain protein, binds to alpha-actinin-1and associates with actin filaments and stress fibers in activated platelets and endothelial cells

A 38-kd protein that associates with F-actin structures in activated platel ets and endothelial cells was purified, cloned, and characterized. The prot ein contains an N-terminal PDZ motif, a large intervening sequence, and a C -terminal LIM domain and was identified as the human homolog of rat CLP36, The study showed that CLP36 associates with actin filaments and stress fibe rs that are formed during shape change and spreading of platelets and durin g migration and contraction of endothelial cells. CLP36 binds to alpha -act inin-1. as shown by coimmunoprecipitation, pull-down experiments, yeast 2-h ybrid analysis, and blot overlay assays and colocalizes with alpha -actinin -1 along endothelial actin stress fibers. In contrast to alpha -actinin-1, CLP36 was absent from focal adhesions in both activated platelets and endot helial cells. The N-terminal part of CLP36 containing the PDZ domain and th e intervening region, but not the LIM domain, targeted enhanced green fluor escent protein fusion proteins to stress fibers in endothelial cells. Yeast 2-hybrid analysis demonstrated that the intervening sequence, but not the PDZ or the LIM domain of CLP36, binds to the spectrinlike repeats 2 and 3 o f alpha -actinin-1. The study further shows that CLP36 binds to alpha -acti nin in resting platelets and translocates as a CLP36/alpha -actinin complex to the newly formed actin cytoskeleton in activated platelets. The results indicate that CLP36 binds via alpha -actinin-1 to actin filaments and stre ss fibers in activated human platelets and endothelial cells. The study sug gests that CLP36 may direct alpha -actinin-1 to specific actin structures a nd at this position might modulate the function of alpha -actinin-1. (C) 20 00 by The American Society of Hematology.