p600, a unique protein required for membrane morphogenesis and cell survival

Abstract

In this article, we identify and characterize p600, a unique 600-kDa retinoblastoma protein- and calmodulin-binding protein. In the nucleus, p600 and retinoblastoma protein seem to act as a chromatin scaffold. In the cytoplasm, p600 and clathrin form a meshwork structure, which could contribute to cytoskeletal organization and membrane morphogenesis. Reduced expression of p600 with interference RNA abrogates integrin-mediated ruffled membrane formation and, furthermore, prevents activation of integrin-mediated survival pathways. Consequently, knockdown of p600 sensitizes cells to apoptosis induced by cell detachment. These findings provide mechanistic insight into the regulation of membrane-proximal events in tumorigenesis.

Fig. 1.

Cloning and mapping p600. (A) Silver staining of the purified RB complex. e-RB was purified from e-RB-expressing HeLa cells by immunoprecipitation with anti-FLAG antibody (lane 2). As a control, mock purification was performed with nontransduced HeLa cells (lane 1). (B) p600 is a calmodulin-binding protein. The purified RB complex (lanes 2 and 4) and nontransduced control (lanes 1 and 3) were incubated with biotinylated calmodulin in buffer containing CaCl₂ (lanes 1 and 2) or EGTA (lanes 3 and 4).

Fig. 2.

Immunolocalization of p600 and RB. (A) Localization of p600, F-actin, and microtubules (MT). In the cytoplasm, p600 is accumulated near the leading edge of protrusion sites (see arrow). (B) Localization of p600, RB, and DNA. The colors detecting stained materials are shown above the images. (Scale bars, 5 μm.)

Fig. 3.

Immunolocalization of p600 in the cytoplasm. (A) Localization of p600 and clathrin. (B) Localization of p600 and calmodulin. (C) Localization of p600 and IP3R. (D) Localization of p600, F-actin, and microtubules (MT). The newly developed surface extension is shown by the arrow. The colors detecting stained materials are shown above the images. (Scale bars, 5 μm.)

Fig. 4.

Knockdown (KD) of p600 causes deficiencies in ruffle formation. (A) Scanning electron microscopic images of the control (Upper) and the p600 knockdown (Lower) cells 20 min after plating on fibronectin-coated plates. (Scale bar, 20 μm.) (B) Immunolocalization of p600 (green) and fibrous actin (red). The image is represented as a stereographic view. (Scale bar, 10 μm.)

Fig. 5.

Knockdown (KD) of p600 sensitizes cells to apoptosis. (A) Stimulation of apoptosis by p600 knockdown. Percentage of apoptotic cells induced during cell proliferation and serum starvation are shown in the control (black) and p600 knockdown (white) bars. Error bars are SDs. (B) Morphological changes induced by p600 knockdown. Scanning electron microscopic images of growing (Left), confluent (Center), and serum-starved (Right) states of the control (Upper) and p600 knockdown fibroblasts (Lower) are shown. Note that most apoptotic cells were removed during sample preparation because apoptosis was induced mostly after detachment from the extracellular matrix. (Scale bar, 20 μm.)

Fig. 6.

Involvement of p600 in integrin-FAK signaling. (A) Analysis of FAK phosphorylation by Western blotting. Cell lysates prepared from growing (lanes 1 and 2) and confluent (lanes 3 and 4) control (lanes 1 and 3) and p600 knockdown (KD) cells (lanes 2 and 4) were analyzed by Western blotting with the indicated antibodies. (B) Analysis of FAK phosphorylation by immunofluorescent microscopy. The control (Left) and p600 knockdown (Right) cells were costained with FAK (green) and phospho-FAK^Y397 (red) antibodies. (Scale bars, 10 μm.)