The RNA-binding protein SERBP1 interacts selectively with the signaling protein RACK1

Abstract

The RACK1 protein interacts with numerous proteins involved in signal transduction, the cytoskeleton, and mRNA splicing and translation. We used the 2-hybrid system to identify additional proteins interacting with RACK1 and isolated the RNA-binding protein SERBP1. SERPB1 shares amino acid sequence homology with HABP4 (also known as Ki-1/57), a component of the RNA spicing machinery that has been shown previously to interact with RACK1. Several different isoforms of SERBP1, generated by alternative mRNA splicing, interacted with RACK1 with indistinguishable interaction strength, as determined by a 2-hybrid beta-galactosidase assay. Analysis of deletion constructs of SERBP1 showed that the C-terminal third of the SERBP1 protein, which contains one of its two substrate sites for protein arginine N-methyltransferase 1 (PRMT1), is necessary and sufficient for it to interact with RACK1. Analysis of single amino acid substitutions in RACK1, identified in a reverse 2-hybrid screen, showed very substantial overlap with those implicated in the interaction of RACK1 with the cAMP-selective phosphodiesterase PDE4D5. These data are consistent with SERBP1 interacting selectively with RACK1, mediated by an extensive interaction surface on both proteins.

Keywords: 2-Hybrid; HABP4; PDE4; PDE4D5; RACK1; SERBP1.

Fig. 1. SERBP1 protein isoforms and key functional features

1a. Amino acid sequence of the longest known (474-amino acid) human SERBP1 isoform. The single-letter amino acid code is used. Regions that are encoded by alternatively-spliced exons and therefore not present in some of the shorter isoforms are shown in gold (i.e., the 63-amino-acid N-terminal region), red, blue or green (see Fig. 1b for more details). The two regions that are targets for arginine methylation by PRMT1 (typically, repeats of sequence RGG or RGR) are shown in grey. The C-terminal region required for interaction with RACK1 is underlined. 1b. Human SERBP1 variants. BLAST searches of amino acid sequence data in GenBank using the 474-amino acid SERBP1 isoform as a query yielded 9 additional isoforms, all encoded by SERBP1 and generated by alternative mRNA splicing. Each of these isoforms differs from the 474-amino acid isoform in that it lacks specific blocks of sequence, as shown.

Fig. 1. SERBP1 protein isoforms and key functional features

1a. Amino acid sequence of the longest known (474-amino acid) human SERBP1 isoform. The single-letter amino acid code is used. Regions that are encoded by alternatively-spliced exons and therefore not present in some of the shorter isoforms are shown in gold (i.e., the 63-amino-acid N-terminal region), red, blue or green (see Fig. 1b for more details). The two regions that are targets for arginine methylation by PRMT1 (typically, repeats of sequence RGG or RGR) are shown in grey. The C-terminal region required for interaction with RACK1 is underlined. 1b. Human SERBP1 variants. BLAST searches of amino acid sequence data in GenBank using the 474-amino acid SERBP1 isoform as a query yielded 9 additional isoforms, all encoded by SERBP1 and generated by alternative mRNA splicing. Each of these isoforms differs from the 474-amino acid isoform in that it lacks specific blocks of sequence, as shown.

Fig. 2. Determination of the region of SERBP1 required for it to interact with RACK1

Yeast 2-hybrid experiments were performed using RACK1 as bait and various regions of the SERBP1 cDNA as prey. For bait, RACK1 was expressed in S. cerevisiae L40 as a LexA fusion (i.e., as pLexARACK1). For prey, the SERBP1 clones were expressed as GAL4 fusions (i.e., in pGADN containing full-length SERBP1, or various deletion subclones thereof). The strength of the Interactions were determined by a filter β-galactosidase assay. Interactions that were indistinguishable from that of RACK1 and full-length SERBP1 were scored as positive (symbol to the right of each construct); those that were indistinguishable from the corresponding empty vectors were scored as negative. All data were obtained in a least 3 independent experiments, using separate transformations. Discrete regions of amino acid sequence encoded by each construct are shown, using the same color scheme as in Fig. 1a.

Fig. 3. Alignment of the amino acid sequence of the long SERBP1 isoform with that of the longest known HABP4 isoform

BLAST searches of human amino acid sequence data in GenBank using the 413-amino acid HABP1 isoform as a query yielded at least 2 additional isoforms, all encoded by HABP4 and generated by alternative mRNA splicing. The longest HABP4 isoform (413 amino acids) was then aligned with the longest SERBP1 isoform (474 amino acids) using BLASTP at www.ncbi.nlm.nih.gov/blast/. The alignment is shown using the single letter amino acid code. Dashes indicate gaps inserted by the alignment program, amino acid identities are indicated by the presence of that amino acid in the line between the 2 sequences, and related amino acids are indicated by the (+) sign. The region of SERBP1 essential for its interaction with RACK1 (amino acids 354 to 474, Fig. 2) is underlined.

Fig. 4. Analysis of mutations in RACK1 that attenuate its interaction with SERBP1 and PDE4D5

Various mutations in RACK1 that attenuated its interaction with PDE4D5 were created by random mutagenesis and identified in a reverse 2-hybrid screen, as we have described previously (12). These mutations were then tested for their ability to attenuate the interaction of RACK1 with SERBP1, as described in Materials and Methods. Yeast S. cerevisiae L40 cells in any given row contained the same bait and those in any given column contained the same prey. Positive interactions, assessed with a filter β-galactosidase assay, produce blue patches, while negative interactions produce amber patches. Controls are vectors alone. Standards are the oncoproteins RAS^V12 and RAF1. This figure shows data typical of experiments performed at least 3 times.