SUPPLEMENTARY INFORMATION Nikandrova et al., 2009 Alternative splicing of SAP97

We identified variants of the rat SAP97 U5 domain containing I2, I3 and/or I5 inserts, as well as full length and “short” variants of the I4 insert, in total rat hippocampus RNA pools isolated at postnatal day 21. The short and long forms of the I4 insert can be explained by the presence of alternative splicing acceptor sites. Splicing acceptor sites are defined by nucleotide sequences in the pre-mRNA that conform to a YYYYYYYYNCAG consensus, where Y stands for either C or T) (1). There are two loose consensus splicing acceptor sites in appropriate positions of SAP97 pre-mRNA: 1. In the intron preceding the I4 exon (TCAAAAATGAAAG), resulting in a full length I4 insert. 2. Within the previously identified full coding sequence of the I4 exon (TCTTGATTACAG), resulting in the I4s variant, which lacks 5 amino acids (Leu-Ile-Leu-Ile-Thr) from the N-terminal segment of the full length I4 insert, and also changes a Asp to His (Fig. 5 shows the sequences). Similar tandem mRNA splicing events are emerging as a common mechanism for increasing the functional diversity of proteins expressed from a single gene (1). Detailed experimental procedures DNA constructs - The myc-SAP97 plasmid was used as a PCR template to amplify full-length SAP97, and fragments encoding N-terminal region (L27; amino acids 1-190), PDZ1/2 (191-375), PDZ3 (426-519) domains and the SH3-GK domains (542-894). The nucleotide sequence of full-length SAP97 was inserted into pFLAG-CMV2 (Sigma) expression vector at EcoRI and BglII sites. Fragments were inserted into pGEX-2T or pGEX-4T (for SH3-GK) at BamHI/EcoRI or EcoRI/XhoI sites, respectively, for bacterial expression of GST-fused proteins. For expression in eukaryotic cells the fragment encoding residues 542-894 (SAP97ΔN) was inserted into pEYFP-Mem vector (Clontech Laboratories, Inc) between EcoR1 and SmaI restriction sites. The FLAG-SAP97 cDNA was digested with BstEII to create a 5’ fragment encoding residues 1-595, designated SAP97ΔC.

The I3 insert was deleted from the GST-SH3-I3I5-GK protein using an overlapping PCR protocol (2). Briefly, the first round of PCR amplified two fragments using pGEX-4T-SH3-I3I5-GK construct as the template. The first fragment encoded the SH3 domain and the N-terminal portion of U5 region up to the beginning of the I3 insert, along with the first 17 (5’) base pairs encoding the I5 insert. The second fragment encoded sequence starting at the N-terminal end of the I5 insert (following the I3 insert) through the C-terminal end of the GK domain. The second round of PCR was performed using a mixture of the two products from the first round reactions as templates with additional primers designed against the 5’ end of SH3 and the 3’ end of the GK domain sequences. The final PCR product was inserted into pGEX-4T vector at EcoRI and XhoI sites after digestion, thus encoding a GST-SH3-I5-GK protein.

To express the SAP97 U5 domain alone with or without the I3 insert, pGEX-4T constructs encoding SH3-I3I5-GK and SH3-I5-GK were used as PCR templates with primers designed against the 5’ end of SH3 (forward) and the 3’ end of GK (reverse) encoding sequences. The PCR product was inserted into pGEX-2T vector at EcoRI and BamHI sites.

Mutations of Ser39 and/or Ser232 to Ala in FLAG-tagged SAP97, and of Thr286 to Asp in CaMKIIα were created by site-directed mutagenesis.

Expression of GST-NR2B (1260-1339, S1303A) was described previously (3). Identification of rat brain SAP97 variants by reverse transcription PCR (RT-PCR) - Total RNA was isolated from postnatal day 21 (P21) rat brain using Trizol reagent (Life Technologies, Inc., Gaithensburg, MD, USA), as described previously (4,5). Briefly, RNA (3 µg) was incubated at 42°C for 1 hr with oligo-dT and MMLV reverse transcriptase (Promega, Madison, WI, USA) in the presence of dNTP (final volume of 50 µl). The resulting cDNA was used as a template for

amplification with Tfl DNA polymerase and primers (50 pmole) designed against 5’ end of SH3 domain (forward) and the 3’ end of GK domain (reverse). PCR products were separated on an agarose gel and visualized by ethidium bromide staining. DNA fragments of the expected size (400-500 bp) were purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA), ligated into pGEMT-Easy vector (Promega) and transformed into E.coli (DH5α). Individual bacterial colonies were screened for the presence of an appropriately sized insert by PCR amplification with primers designed against vector (sp6 and T7 promoters), and then with the original SAP97-specific primers used for the RT-PCR reaction. The SAP97 positive plasmids were sequenced to identify U5 splice variants (see Results). The four variant sequences have been deposited in the GenBank database with Accession Numbers 1229146, 1228740, 1226960, and 1226950.

In order to generate DNA constructs encoding GST-fused SH3-U5-GK proteins containing differentially spliced U5 region, selected RT-PCR clones were mixed with a cDNA fragment encoding GK domain alone and primers designed against the 5’ end of SH3 and the 3’ end of GK domain. After PCR amplification, products encoding the entire SH3-U5-GK domains of SAP97 were inserted into pGEX-4T vector at EcoRI/XhoI sites. Full-length FLAG-tagged SAP97 variants were generated by excising fragments encoding U5 region variants from relevant pGEX-4T constructs using BamHI and BstEII, and inserting them between BamHI and BstEII sites in the original FLAG-tagged SAP97 construct. All constructs were verified by sequencing. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) with HEK293 cells mRNA - Total RNA was harvested from HEK293 cells using the RNEasy kit (Qiagen; Valencia, CA) following the manufacturer’s protocol. A one-step RT-PCR (Qiagen) reaction was performed using ≈0.5 µg of total RNA. Splice form specific primers corresponding to sequences encoding the human SAP97 (hDLG1)-I2 (nucleotides 2224-2204: 5’-GCCTTTTGATCCCATGTCGTC-3’) and -I3 (nucleotides 2227-2203: 5’-GAGGTTCTTTTTACGCTTGTCATTG-3’) inserts were used in conjunction with a forward primer corresponding to nucleotides 1937-1957 of hDLG1 (5’-TCCCTCTATGTCAGAGCCCTT-3’) that is common to both variants. All primers shared 100% identity with the corresponding sequences in rat allowing for semi-quantitative comparison with parallel PCR reactions using rat isoform cDNAs as templates (≈10 ng each). RT-PCR reactions were carried out on a thermocycler (Techne; Cambridge, UK) using the following protocol: 95 ºC for 2 min for initial denaturation, 50 ºC for 30 min for reverse transcription, 95 ºC for 15 min to inactivate the reverse transcriptase. The subsequent PCR was carried out for 35 cycles. Each cycle consisted of 1 min denaturing at 94 ºC, 1 min annealing at 50 ºC, and 1 min extension at 72 ºC. The PCR of rat cloned I2 and I3 containing splice variants was carried out for 30 cycles using the same parameters. The resulting PCR products were resolved on 2.5 % agarose gels. Gels were stained with ethidium bromide and visualized using a Alpha Imager EP (Alpha Innotech; San Leandro, CA). Protein purification – GST fusion proteins were expressed from pGEX-2T or -4T constructs in E.coli (BL21). After transformed cell cultures reached OD≥0.750 (λ=600 nm) protein expression was induced using 1 mM IPTG for 4 hours. Cells were lysed and sonicated in lysis buffer (0.5% (v/v) Triton X-100, 50 mM Tris-HCl pH 7.4, 200 mM NaCl, 0.5 mM PMSF, 1 mM benzamidine, 1 mg/ml lysozyme, protease inhibitors cocktail (Sigma, P2714)). GST fusion proteins were isolated on glutathione-agarose (Sigma-Aldrich), washed extensively with washing buffer (0.5% (v/v) Triton X-100, 50 mM Tris-HCl pH 7.4, 200 mM NaCl, 0.5mM EDTA, 0.5 nM EGTA, 0.1 mM PMSF, 0.1 mM benzamidine), and then eluted using 20 mM glutathione in the same buffer adjusted to pH 8.0. Proteins were dialyzed against washing buffer for 16 hrs. Protein concentrations were estimated using the Bradford assay (BioRad, Rockford, IL, USA). Aliquots were stored at -80°C.

CaMKIIα autophosphorylation - CaMKIIα (5 µM) was incubated for 1 min on ice in 50 mM HEPES pH 7.5, 2 mM magnesium acetate, 1.5 mM CaCl2, 10 µM calmodulin, 20 µM [γ-32P]ATP (20,000 cpm/pmol). Nonradioactive ATP (2 mM final) was added, followed by an additional incubation for 1 min on ice. The reaction was stopped by the addition of EDTA (10 mM), NaF (25 mM), NaCl (0.5 M), and Tween-20 (0.1% v/v). After incubating for 1 hr on ice, the 32P-labeled CaMKIIα was desalted on Sephadex G-50 column (15 ml) equilibrated with 50 mM Tris-HCl pH 7.4, containing 0.5 M NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% (v/v) Tween 20. Peak radioactive fractions (0.5 ml) were pooled and aliquots of the final pool and the reaction mix were spotted on P81 phosphocellulose papers (Whatman) and washed with water to determine the autophosphorylation stoichiometry and the concentration of 32P-labeled CaMKIIα in the pool by liquid scintillation counting. Immunoblotting - Proteins were resolved by SDS-PAGE under reducing conditions and transferred to nitrocellulose membrane in 10 mM N-cyclohexyl-3-aminopropanesulfonic acid buffer. After blocking with 5% Carnation non-fat milk, or 0.1% BSA (for FLAG immunoblot) in TBST buffer (50 mM Tris-HCl pH 7.4, 140 mM NaCl, 0.1 % (v/v) Tween 20) for 1 hr at 20-25°C, membranes were incubated overnight at 4°C with primary antibodies diluted in TBST with 5% milk or 0.1% BSA. Membranes were washed 3 times in TBST buffer, and incubated for 2 hrs at 20-25°C with secondary antibodies diluted in TBST with 5% milk or 0.1% BSA. After extensive washing in TBST, immunobloted proteins were visualized using Enhanced Chemiluminescence Substrate (ECL) (PerkinElmer, Waltham, MA, USA) following the manufacturer’s protocol.

Legends for Supplementary Figures: Figure S1. Selective association of CaMKII with SH3-I3I5-GK domains of SAP97 in an overlay assay. Indicated amounts of each GST-fusion protein in 500 µl TBS (50 mM Tris-HCl pH 7.5, 140 mM NaCl) were directly applied to a nitrocellulose membrane using a slot blotter (Schleicher & Shuell, Keene, NH, USA). The membrane was incubated for 1 hr in blocking buffer (50 mM Tris-HCl, pH 7.5, 140 mM NaCl, 3% (v/v) Tween-20, and 5% (w/v) milk), and then for 2 hrs with [32P-Thr286]CaMKII (see Materials and Methods) (63 nM) in incubation buffer (50 mM Tris-HCl, pH 7.5, 140 mM NaCl, 0.1% Tween-20, and 5% milk). After washing 5 times in incubation buffer, bound CaMKII was visualized by autoradiography (bottom panel). Immobilized GST-proteins were subsequently detected by immunoblot with a horseradish peroxidase-conjugated anti-GST antibody (Amersham, Piscataway, NJ, USA) (1:1000) (top panel). These data are representative of 3 similar experiments. Figure S2. CaMKII phosphorylation in the C-terminal domains of SAP97 is significantly reduced by deletion of the I3 insert. The indicated GST fusion proteins, with or without the I3 insert (1 µM each) were phosphorylated by CaMKII in vitro (see Materials and Methods). Surprisingly, the total stoichiometry for phosphorylation of GST-U5-I3I5 was about 3 times higher than that of GST-SH3-I3I5-GK. This may be because the U5 domain is relatively unfolded in the shorter construct so that more Ser/Thr residues are readily available for phosphorylation. The top panels show representative autoradiographs, whereas bar graphs plot phosphorylation in proteins lacking the I3 insert relative to the control protein containing I3 (mean±sem) from n=7 or n=5 experiments for GST-SH3-U5-GK and GST-U5 proteins, respectively (*: p<0.005).

Figure S3. Similarities and differences in CaMKII interactions with NR2B and SAP97. A. GST-SH3-I3I5-GK or GST-NR2B(1290-1309) (125 pmole each) were immobilized on nitrocellulose membrane (see Fig. S1), and then incubated with [32P-Thr286]CaMKII (30 nM) in the absence or presence of the indicated synthetic peptides (100 µM each). Membranes were exposed to X-ray film, and a representative image from three independent experiments is shown. Binding was quantified and normalized to CaMKII binding in the presence of syntide-2: mean±SEM binding is plotted. An ANOVA showed significant variation in each data set. Post-testing revealed p<0.05 vs hypothetical value of 1 where indicated (*). Autocamtide-2 (AC-2) and syntide-2 are commonly used CaMKII substrate peptides that interact with distinct portions of the catalytic domain (T-site and S-site, respectively (6-8)). The WT NR2B peptide is an analog of the CaMKII binding domain in NR2B (residues 1290-1309) and is thought to interact at the T site of CaMKII: incorporation of the R1299N mutation substantially reduces CaMKII binding to NR2B (9). (NR2B peptides were custom synthesized by EZBiolabs.). The data suggest that binding sites for NR2B and SAP97 overlap the T site in the CaMKII catalytic domain. B. GST co-sedimentation assay of non-phosphorylated CaMKII with SH3GK under various conditions. GST, GST-SH3-GK or GST-NR2B (1260-1309) were incubated with purified non-phosphorylated CaMKIIα in GST-co-sedimentation assay (as described in “Materials and Methods”) in the presence of ADP (100 µM) and/or Ca2+/calmodulin (1 mM/500 nM). Protein complexes were resolved by SDS-PAGE and co-precipitated CaMKII was detected with specific antibody. The combined addition of Ca2+/calmodulin and ADP strongly stimulates binding of non-phosphorylated CaMKII to NR2B (see (10)), but not to SAP97. Figure S4. SAP97(I3) and SAP97(I2) splicing variants are present in HEK293. Representative agarose gel with products of PCR performed with pair of primers and templates as indicated: forward primer 1 was designed against a region common to both variants, reverse primers 2 and 3 were designed against exons encoding the I2 and I3 inserts, respectively. References for Supplementary Information 1. Hiller, M., and Platzer, M. (2008) Trends Genet 24, 246-255 2. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Gene 77, 61-68 3. Strack, S., and Colbran, R. J. (1998) J Biol Chem 273, 20689-20692 4. Jiao, Y., Robison, A. J., Bass, M. A., and Colbran, R. J. (2008) J Neurochem 105, 1746-1760 5. Strack, S., Robison, A. J., Bass, M. A., and Colbran, R. J. (2000) J Biol Chem 275, 25061-

25064 6. Ishida, A., Kitani, T., Okuno, S., and Fujisawa, H. (1994) J Biochem 115, 1075-1082 7. Lisman, J. E., and McIntyre, C. C. (2001) Curr Biol 11, R788-791 8. Bayer, K. U., LeBel, E., McDonald, G. L., O'Leary, H., Schulman, H., and De Koninck, P.

(2006) J Neurosci 26, 1164-1174 9. Strack, S., McNeill, R. B., and Colbran, R. J. (2000) J Biol Chem 275, 23798-23806 10. Robison, A. J., Bartlett, R. K., Bass, M. A., and Colbran, R. J. (2005) J Biol Chem 280,

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Supplementary Figure 1.

GST PDZ1-2 PDZ3 SH3GK

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adio

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Supplementary Figure 2.

SH3-U5-GK U5I5 I3I5 I5 I3I5

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Autoradiography

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Supplementary figure 3.

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pCMV-FlagSAP97(I3I5) HEK293 cells

PCR RT-PCR

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Supplementary Figure 4