Biochemical interaction of an actin‐capping protein, CapZ, with NAP‐22

Abstract

NAP-22 is a neuronal protein localized in the presynaptic membrane and synaptic vesicles and recovered in a Triton-insoluble low-density microdomain fraction after biochemical fractionation of the synaptic plasma membrane. NAP-22 organizes membrane microdomains through binding to membrane lipids such as cholesterol, phosphatidylethanolamine, and phosphatidylinositol 4,5-bisphosphate. In this study, NAP-22-binding proteins were screened through the pull-down assay using brain-derived NAP-22 bound to Sepharose 4B. An actin-capping protein, CapZ, was identified in the precipitate through mass spectrometry and Western blotting. CapZ was then expressed in E. coli and the purified protein-bound NAP-22 directly. Because bacterially expressed NAP-22 bound CapZ, it was determined that the N-terminal myristoyl moiety of NAP-22 is not necessary for the binding. The binding of NAP-22 showed no effect on the actin nucleation activity of CapZ measured with centrifugation and viscometric assays. Hence, the CapZ-NAP-22 complex could work as the nucleation site of actin polymerization or as the actin filament-anchoring site on the membrane microdomain.

Repression of Transcription by WT1-BASP1 Requires the Myristoylation of BASP1 and the PIP2-Dependent Recruitment of Histone Deacetylase

“…In contrast, and consistent with the fact that BASP1 G2A localizes to the promoter regions of WT1-responsive genes (Figure 3D), we found that anti-WT1 antibodies coimmunoprecipitated both wild-type BASP1 and BASP1 G2A (Figure 4D). The actin-capping protein CapZ was previously shown to bind to BASP1 independently of myristoylation status (Odagaki et al, 2009), and consistent with this we found that CapZ antibodies coimmunoprecipitated both wild-type BASP1 and BASP1 G2A (Figure 4D, lower panel). Taken together, the data in Figure 4D suggest that WT1 interacts with HDAC1 through myristoylation of BASP1 and that BASP1 G2A is specifically defective in interacting with HDAC1.…”

Oligomeric structure of brain abundant proteins GAP-43 and BASP1

“…According to the first model, this regulation may be accomplished by their direct interaction with actin or actin-binding proteins (He et al, 1997;Odagaki et al, 2009). The second model presumes that the proteins can bind electrostatically (by means of their basic EDs) and thereby sequester anionic phosphatidylinositol-4,5-diphosphate (PIP 2 ) in the inner leaflet of the plasma membrane Wang et al, 2002;Epand et al, 2004;Tong et al, 2008).…”

Tight binding of NAP-22 with acidic membrane lipids

“…Addition of CHL to the mixture of C-NAP-22 and PS showed little effect on the mobility of NAP-22 in CN-PAGE (data not shown) and electron microscopic observation of Tritontreated NAP-22/PS/CHL complex showed no clear image, molecular background of the Triton-insolubility of the complex is unclear at present. Since, many proteins are known to interact with acidic phospholipids, studies of the effect of NAP-22 on these proteins through the binding to acidic phospholipids will be important to understand the molecular interactions at the inner leaflet of raft [11,13,15,19,20,22].…”

“…Cholesterol distribution and the localization of this protein were hence considered intimately related [10]. In order to elucidate additional functions of NAP-22, a screening of NAP-22-binding proteins was performed using NAP-22-coupled Sepharose beads and several proteins such as an actin binding protein (CapZ␣) [15], synaptojanin [21], and glutamate decarboxylase (GAD) [12] were identified. Other functions of NAP-22 in transcriptional regulation, apoptosis, and ion channel formation were also reported by other groups [3,16,17].…”