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

Abstract

A 38-kd protein that associates with F-actin structures in activated platelets and endothelial cells was purified, cloned, and characterized. The protein 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 fibers that are formed during shape change and spreading of platelets and during migration and contraction of endothelial cells. CLP36 binds to α-actinin-1 as shown by coimmunoprecipitation, pull-down experiments, yeast 2-hybrid analysis, and blot overlay assays and colocalizes with α-actinin-1 along endothelial actin stress fibers. In contrast to α-actinin-1, CLP36 was absent from focal adhesions in both activated platelets and endothelial cells. The N-terminal part of CLP36 containing the PDZ domain and the intervening region, but not the LIM domain, targeted enhanced green fluorescent 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 of α-actinin-1. The study further shows that CLP36 binds to α-actinin in resting platelets and translocates as a CLP36/α-actinin complex to the newly formed actin cytoskeleton in activated platelets. The results indicate that CLP36 binds via α-actinin-1 to actin filaments and stress fibers in activated human platelets and endothelial cells. The study suggests that CLP36 may direct α-actinin-1 to specific actin structures and at this position might modulate the function of α-actinin-1.