Direct interaction between caldesmon and cortactin

Abstract

Actin polymerization and depolymerization plays a central role in controlling a wide spectrum of cellular processes. There are many actin-binding proteins in eukaryotic cells. Their roles in the remodeling of the actin architecture and whether they work cooperatively await further study. Caldesmon (CaD) is an actin-binding protein present in nearly all mammalian cells. Cortactin is another actin-binding protein found mainly in the cell cortex. There have been no reports suggesting that CaD and cortactin interact with each other or work as partners. Here, we present evidence that CaD binds cortactin directly by overlay, pull-down assays, ELISA, and by column chromatography. The interaction involves the N-terminal region of cortactin and the C-terminal region of CaD, and appears to be enhanced by divalent metal ions. Cortactin competes with both full-length CaD and its C-terminal fragment for actin binding. Binding of cortactin partially alleviates the inhibitory effect of CaD on the actomyosin ATPase activity. Not only can binding be demonstrated in vitro, the two proteins also co-localize in activated cells at the cortex. Whether such interactions bear any functional significance awaits further investigation.

Fig. 1

Co-localization of CaD (red) and cortactin (green) in the lamellipodium and ruffles of newly divided and spreading rat aorta fibroblasts. The right panels show the merged image. In these cells CaD is also phosphorylated at ERK sites (Kordowska et al., 2006). For CaD staining, anti-CaD and anti-pS789CaD antibodies were used in the upper and lower panels, respectively. Scale bars: 50 μm.

Fig. 2

Overlay assays of recombinant cortactin-B blotted with full-length chicken gizzard CaD (Lanes 1 and 1’), CaD fragment H32K (Lanes 2 and 2’) or N240 (Lanes 3 and 3’) in the presence (Lanes 1–3) or absence (Lanes 1’–3’) of Ca²⁺. Bound CaD peptides were detected by anti-CaD antibody and visualized by Odyssey infrared imaging. M and M’: molecular weight markers.

Fig. 3

Pull-down assay results of cortactin-Sepharose beads reacted with the N240 (Lanes 1 and 5), H32K (Lanes 2 and 4) and chicken gizzard CaD (Lanes 3 and 6) in the presence (Lanes 1–3) and absence (Lanes 4–6) of Ca²⁺. M: molecular weight markers.

Fig. 4

Cortactin is retained to the H32K-affinity column. (A) H32K was first coupled with CNBr-activated sepharose 4B beads. Crude cell extract containing recombinant cortactin-B was loaded to the resulting H32K-Sepharose affinity column in the presence of 1 mM Ca²⁺, and eluted with 10 mM EDTA. M: molecular weight standard; Lane 1: cell extract containing over-expressed cortactin-B; Lane 2: eluent after washing; Lane 3–6: fractions eluted with 10 mM EDTA, 20 mM Tris-Cl pH7.5, 100 mM NaCl; Lane 7–10: fractions eluted with 50 mM glycine pH2.3, 250 mM NaCl. (B): Western blotting result by Odyssey of all cortactin-containing fractions eluted from the H32K-Sepharose column. The fractions in Panel A were transferred onto PVDF membrane and incubated with mouse anti-cortactin monoclonal antibody followed by fluorescently labeled anti-mouse secondary antibody. All lanes correspond to the lanes of the same number in Panel A.

Fig. 4

Cortactin is retained to the H32K-affinity column. (A) H32K was first coupled with CNBr-activated sepharose 4B beads. Crude cell extract containing recombinant cortactin-B was loaded to the resulting H32K-Sepharose affinity column in the presence of 1 mM Ca²⁺, and eluted with 10 mM EDTA. M: molecular weight standard; Lane 1: cell extract containing over-expressed cortactin-B; Lane 2: eluent after washing; Lane 3–6: fractions eluted with 10 mM EDTA, 20 mM Tris-Cl pH7.5, 100 mM NaCl; Lane 7–10: fractions eluted with 50 mM glycine pH2.3, 250 mM NaCl. (B): Western blotting result by Odyssey of all cortactin-containing fractions eluted from the H32K-Sepharose column. The fractions in Panel A were transferred onto PVDF membrane and incubated with mouse anti-cortactin monoclonal antibody followed by fluorescently labeled anti-mouse secondary antibody. All lanes correspond to the lanes of the same number in Panel A.

Fig. 5

ELISA results showing cortactin interacts with both full-length gizzard CaD (squares, dash line) and H32K (circles, solid line) in the presence of Ca²⁺. The 96-well plate was coated with cortactin-B. After incubated with different ratio of h-CaD or H32K, followed with polyclonal anti-CaD antibodies (H-C) and anti-rabbit secondary antibody, the OD of each well was measured at 490 nm.

Fig. 6

Competition between cortactin and H32K for F-actin binding. (A) Cortactin-B (1 μM) or H32K (2 μM), or both, mixed with F-actin (6 μM) in F-buffer, then centrifuged at 80K rpm for 20 min. Both the supernatant (Lanes 2, 4, 6) and pellet (Lanes 3, 5, 7) fractions were run through 10% SDS-PAGE after washing. M: molecular weight standard; Lane 1: cortactin-B; Lanes 2 and 3: cortactin-B and actin; Lanes 4 and 5: H32K and actin; Lanes 6 and 7: cortactin, H32K and actin. (B) Cortactin-B mixed with F-actin and H32K in F-buffer, then centrifuged at 80K rpm for 20 min, the pellets were run through 10% SDS-PAGE after washing. M: molecular weight standard; Lane 1: H32K; Lane 2: cortactin-B; Lane3: actin; Lane 4: 1 μM cortactin-B and 3 μM actin; Lane 5: 1 μM cortactin-B mixed with 3 μM actin and then added 5 μM H32K; Lane 6: 5 μM H32K mixed with 3 μM actin; Lane 7: 1 μM H32K mixed with 3 μM actin, and then added 5 μM cortactin-B.

Fig. 6

Competition between cortactin and H32K for F-actin binding. (A) Cortactin-B (1 μM) or H32K (2 μM), or both, mixed with F-actin (6 μM) in F-buffer, then centrifuged at 80K rpm for 20 min. Both the supernatant (Lanes 2, 4, 6) and pellet (Lanes 3, 5, 7) fractions were run through 10% SDS-PAGE after washing. M: molecular weight standard; Lane 1: cortactin-B; Lanes 2 and 3: cortactin-B and actin; Lanes 4 and 5: H32K and actin; Lanes 6 and 7: cortactin, H32K and actin. (B) Cortactin-B mixed with F-actin and H32K in F-buffer, then centrifuged at 80K rpm for 20 min, the pellets were run through 10% SDS-PAGE after washing. M: molecular weight standard; Lane 1: H32K; Lane 2: cortactin-B; Lane3: actin; Lane 4: 1 μM cortactin-B and 3 μM actin; Lane 5: 1 μM cortactin-B mixed with 3 μM actin and then added 5 μM H32K; Lane 6: 5 μM H32K mixed with 3 μM actin; Lane 7: 1 μM H32K mixed with 3 μM actin, and then added 5 μM cortactin-B.