Scanning the human proteome for calmodulin-binding proteins

Abstract

The calcium ion (Ca(2+)) is a ubiquitous second messenger that is crucial for the regulation of a wide variety of cellular processes. The diverse transient signals transduced by Ca(2+) are mediated by intracellular Ca(2+)-binding proteins, also known as Ca(2+) sensors. A key obstacle to studying many Ca(2+)-sensing proteins is the difficulty in identifying the numerous downstream target interactions that respond to Ca(2+)-induced conformational changes. Among a number of Ca(2+) sensors in the eukaryotic cell, calmodulin (CaM) is the most widespread and the best studied. Employing the mRNA display technique, we have scanned the human proteome for CaM-binding proteins and have identified and characterized a large number of both known and previously uncharacterized proteins that interact with CaM in a Ca(2+)-dependent manner. The interactions of several identified proteins with Ca(2+)/CaM were confirmed by using pull-down assays and coimmunoprecipitation. Many of the CaM-binding proteins identified belong to protein families such as the DEAD/H box proteins, ribosomal proteins, proteasome 26S subunits, and deubiquitinating enzymes, suggesting the possible involvement of Ca(2+)/CaM in different signaling pathways. The selection method described herein could be used to identify the binding partners of other calcium sensors on the proteome-wide scale.

Fig. 1.

Selection of CaM-binding proteins by using an mRNA-displayed human proteome library. mRNA, green; DNA, red; protein, yellow; puromycin, blue circle. SA, streptavidin beads.

Fig. 2.

In vitro binding analysis of selected proteins with biotin-CaM. Full-length proteins or fragments were generated by a transcription/translation (TNT) reaction. An aliquot of the TNT mixture was incubated with an appropriate amount of biotinylated CaM in the presence of 1 mM CaCl₂, followed by mixing with streptavidin-agarose beads. The beads were washed, and the bound molecules were eluted by using a buffer containing 2 mM EGTA. (A) Selected protein fragments, showing expressed protein (C) and eluent (E). The complete fragment binding data corresponding to proteins listed in Table 1 are provided in Fig. 6. The last three samples (N1-N3) are negative controls. N1 (similar to cardiac morphogenesis protein) and N2 (MLL3 protein) are two protein fragments that do not bind to CaM in the binding assays. N3 is a protein fragment from the non-CaM binding region of a known CaM-binding protein (ATP2B1). (B) Full-length proteins, showing expressed protein (C), the flowthrough (F), the last wash (W), and the eluent (E). N4, negative control (KIAA0367 from an irrelevant project). The names of all other proteins are listed in Table 1 according to their IDs.

Fig. 3.

Calcium-dependent interactions between calmodulin and AKT1, CDC5-L, and RAD23B. CaM-Sepharose pull-down assays by using mouse brain lysate (A-C) and human HeLa cell lysate (D-F). Lysates were incubated with CaM-Sepharose 4B beads in the presence of CaCl₂ (Upper) or EGTA (Lower) as described in the experimental section. L, lysate; FT, flowthrough; E, elution; W, wash. (A and D) AKT-1. (B and E) CDC5-L. (C and F) RAD23B. Anti-AKT1 and anti-CaM were from Santa Cruz Biotechnology, anti-CDC5L from BD Biosciences, and anti-RAD23B from Rockland (Gilbertsville, PA).

Fig. 4.

Coimmunoprecipitation from HeLa cell lysate of AKT-1 and CDC5-L with CaM. Equal amounts of precleared HeLa cell lysate were incubated with anti-CaM antibody in the presence of CaM-binding buffer containing either 1 mM CaCl₂ or 2 mM EGTA. The protein complex was captured by protein A/G-agarose conjugate and eluted with buffer C. AKT or CDC5L associated with CaM was detected by Western blot analysis by using anti-AKT (A) or anti-CDC5L (B). L, lysate; FT, flowthrough; E, elution of pellets by using buffer C; W, wash of pellets by using buffer B.