Carboxyl-Terminal Modulator Protein (CTMP), a Negative Regulator of PKB/Akt and v-Akt at the Plasma Membrane Sauveur-Michel Maira, Ivana Galetic, Derek P. Brazil, Stefanie Kaech, Evan Ingley, Marcus Thelen, and Brian A. Hemmings

Supplementary Material

Supplemental Figure 1. Interaction of human PKB with CTMP in the yeast two-hybrid system. (A and B) Domain structure of Gal4 DNA binding constructs. DBD, DNA binding domain. (C) Cotransformants of constructs (a) to (h) in (B) with the empty vector pGAD424 (Gal-AD), CTMP expression vector pGADGH-CTMP (Gal-AD.CTMP), or control vectors for specificity pVA3 (Gal-AD.p53) and pLAM5´ (Gal-AD-hLAM-C) in HF7c were selected on Leu^-/Trp^- plates and then replated onto Leu^-/Trp^-/His^- plates to detect activation of the HIS3 reporter. (C) Liquid -gal assays of cotransformants of constructs (a) to (h) in (B) with pGADGH-CTMP in SFY526 cells. A Bam HI-Eco RI PCR fragment encoding amino acids 147 to 480 of PKB (1) was cloned in the pPC62 vector (a generous gift from D. Nathans, Howard Hughes Medical Institute, Baltimore, MD, USA). This vector was used as "bait" in the two-hybrid screening. Yeast strains SFY526 and PCY2 were used for protein-protein interaction experiments, whereas strain HF7c was used for library screening. Yeast techniques were performed exactly as described in the MATCHMAKER Two-Hybrid System from Clontech, and as described (2).

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Supplemental Figure 2. Expression patterns of the CTMP gene and subcellular localization. (A) RT-PCR was performed on total RNA from the indicated human tissues and HeLa cells, with either CTMP-specific (upper panel, a+b) or interleukin 1 (IL-1)-specific (bottom panel) primers. pBluescript-CTMP (pCTMP) was used as a positive control (3). (B) Immunoprecipitation (IP) and immunoblotting (IB) of crude lysates (500 g) from various cell lines were performed with the CTMP antibody 99390 (14). Clone H is a cell line stably expressing Flag-CTMP (see Fig. 5A). CTMP from this cell line migrated as a doublet. (C) S100 and P100 fractions (31) from HeLa cells were analyzed for CTMP expression. The asterisk (B and C) indicates the position of the IgG light chain.

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Supplemental Figure 3. The human CTMP gene products. (A) The human CTMP gene is composed of at least eight exons, is approximately 40 kb long, and is located on chromosome 1q21. Exon 1 contains the ATG codon, whereas exon 6 contains the stop codon. The gene produces at least two products, CTMP and CTMP-s1. The CTMP cDNA that encodes the full-length CTMP protein is described in Fig. 1 of the report. The CTMP-s1 contains one more exon (2a), that is spliced in, between exons 2 and 3. (B) The CTMP gene products can be detected by RT-PCR using different pairs of primers specific for the different exons. The extra products of the gene exclusively arise from an exon insertion between 2 and 3, because only primers flanking exon 2a give an extra product in the RT-PCR reaction (c + i; a + i; and a + b). Oligomers a and b are as those used in Web fig. 2A (3).

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To view this movie, download a QuickTime viewer.

• Movie loop 1

• Movie loop 2

  This shows a time-lapse microscopy movie showing the localization of transiently expressed GFP-CTMP in a moving NIH 3T3 cell. The cell is moving along a northeast to southwest diagonal. Areas of high fluorescence intensity (i.e., CTMP expression) mainly colocalize with the rapidly moving membrane ruffles at the leading edge of the cell.

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References and Notes
1. M. Andjelkovic et al., J. Biol. Chem. 272, 31515 (1997).
2. F. M. Ausubel et al., Current Protocols in Molecular Biology (Wiley, New York, 1994).
3. Human RNA was purchased from Clontech, with the exception of HeLa cell RNA, which was prepared using the Trizol protocol (Gibco). Reverse-transcription reactions were performed using the GeneAmp RNA PCR kit (PE Biosystems). The oligonucleotides 5´-TCTGAGGAAGTCATTCTTAAG-3´ (a) and 5´-CTCATCAACA CTCTGAACATT-3´ (b) were used to amplify the expected 545-bp CTMP product. Control oligonucleotides 5´-GTCTCTGAATCAGAAATCCTTCTATC-3´ and 5´-CATGTCAAATTTCACTGCTTCATCC-3´ were used to amplify an IL-1-specific sequence of 308 bp present in the synthetic pAW109 RNA added to all reactions to quantify the efficiency of the reverse transcription. PCR amplification was carried out using Taq polymerase (Roche Biochemicals). For the transfected antisense CTMP cDNA (Fig. 4D), oligonucleotides T7 and (b) were used in RT-PCR reactions to distinguish the antisense construct from the endogenous CTMP cDNA.