Identification of non-ser/thr-pro consensus motifs for Cdk1 and their roles in mitotic regulation of C2H2 zinc finger proteins and Ect2

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SUPPLEMENTARY INFORMATION Identification of non-ser/thr-pro consensus motifs for Cdk1 and their roles in mitotic regulation of C2H2 zinc finger proteins and Ect2 Kazuhiro Suzuki, Kosuke Sako, Kazuhiro Akiyama, Michitaka Isoda, Chiharu Senoo, Nobushige Nakajo, and Noriyuki Sagata

a Ikaros (S196) GST WT T194A H195A S196A V197A G198A K199A P200A H201A c I M RO Relative 100 60 68 42 69 93 39 63 78 Flag-IP Flag-YY1 IB: Flag IB: HpTGEKP ( - ) WT ( - ) WT WT IB b GST HpTGEKP -ATP WT T348A +ATP WT T348A 34 34 Input IB: HpTGEKP IB: CycB1 124 64 47 47 Supplementary Figure S1. In vitro Cdk1 kinase assays of Ikaros, recognition of Cdk1-phosphorylated YY1 by α- HpTGEKP, and the effect of RO-3306 on YY1 phosphorylation. (a) GST-fused Ikaros peptides (WT or the indicated Alamutants; residues 184-208) were incubated with cyclin B1-Cdk1 and [γ- ]ATP, and analyzed as in Fig. 1b. (b) GST-fused YY1 peptides (WT or T348A; residues 336-360) were incubated with cyclin B1-Cdk1 in the presence or absence of cold ATP and analyzed by immunoblotting with the indicated antibodies. Note that α-hptgekp specifically recognizes Cdk1- phosphorylated YY1(WT) peptides. (c) Flag/His 6 -tagged YY1(WT) protein was incubated with either interphase (I) or M phase (M) extracts (from Xenopus eggs) in the presence or absence of RO-3306 (RO; 300 µm) and then subjected to Flagimmunoprecipitation followed by immunoblotting with the indicated antibodies. In input, the arrowhead shows Flag/His 6 - tagged YY1(WT).

Cullin 4B NLS 26 GNTSTTPPTSAKKRKLNSSS 45 I M Flag-IP Flag-Cullin 4B-F IB: Flag IB: Importin β ( ) WT KR/AA ( ) WT KR/AA T31A SA T31A/SA 15 Input Imoprtin β binding 1 0.5 0 IB: Importin β IB: CycB1 47 Supplementary Figure S2. Cdk1 inhibition of the NLS-containing non-s/t-p consensus sequence of Cullin 4B. Upper: A sequence spanning the NLS-containing non-s/t-p consensus sequence of (human) Cullin 4B. The mitotic phosphoaccepter Ser of the non-s/t-p consensus sequence is shown in blue, the -2 Pro in green, and the consecutive Arg/Lys residues (NLS) in red. Note that, according to previous work (1), Thr31 followed by a Pro and preceding Ser is also mitotically phosphorylated, perhaps by Cdk1. Lower: Flag/His 6 -tagged Cullin 4B fragments (WT or the indicated Ala-mutants; residues 1-50) were incubated with either interphase (I) or M phase (M) extracts (from Xenopus eggs), and analyzed as in Fig. 5c (mean ± SD, n=4). KR/2A, an NLS mutant. Note that the SA but not T31A mutation can substantially restore the binding of Cullin 4B to Importin β in M phase extracts, indicating that Cullin 4B-Importin β binding is inhibited by mitotic phosphorylation of Ser (but not Thr31).

80 Relative phosphorylation 60 40 20 0 XXAXXXXXX XXSXXXXXX XXSPXXXXX XXSPRXXXX XXSPXRXXX XXSPXXRXX XXSPXXXRX XXSPRRXXX XXSPXRRXX XXSPXXRRX XXSPRRRXX XXSPXRRRX XXSPRRRRX XXSPRRRRR PXSPXXXXX PXSPRXXXX PXSPRRXXX PXSPRRRXX PXSPRRRRX PXSPRRRRR 0.1 0.1 1.0 1.8 2.8 1.6 1.3 23.0 16.1 4.2 43.2 32.3 49.7 55.1 1.8 2.8 34.9 47.0 62.1 69.9 Supplementary Figure S3. In vitro phosphorylation of the conventional S/T-P motif sequences using Arg/Lys-scanning oriented peptide libraries. Schematic representation of Arg/Lys-scanning oriented peptide libraries are shown in Fig. 2a. In each peptide library used (shown only for the -2 to the +6 positions in the figure), R corresponds to R' in Fig. 2a, while X is either X 0, X 1, X 2, X 3, depending on its corresponding positions in Fig. 2a. Each peptide library was incubated with [γ- ]ATP and cyclin B1-Cdk1, and analyzed as described in Materials and Methods. The radioactive signal of each library ( ) was quantitated and its value is shown just above the signal and in the bar diagram, with the value of XXSPXXXXX being set at 1.0 (mean ± SD, n=3).

Figure 1b Figure 1c Figure 1d Figure 1e Figure 1f Figure 1g Supplementary Figure S4. Full scans for images used in Figure 1.

Figure 4b Figure 4e Cdk1 Plk1 Aurora B Figure 4f Figure 4c Flag-IP Flag HpTGEKP 124 Figure 4d Input HpTGEKP 64 47 CycB1 47 Supplementary Figure S5. Full scans for images used in Figure 4.

Figure 5b Flag-IP Flag Importin β Figure 5d 15 124 124 Figure 5c Input Importin β Flag-IP Flag Importin β Importin β 15 124 124 Flag-IP Flag Importin β Figure 5e 15 124 Input 124 CycB1 47 Input Importin β Supplementary Figure S6. Full scans for images used in Figure 5.

Supplementary Table S1. Primers. Vimentin L39A 5'-CGCACCTACAGCGCGGGCAGCGCGCTGCGC-3' 5'-GCGCAGCGCGCTGCCCGCGCTGTAGGTGCG-3' Vimentin G40A 5'-ACCTACAGCCTGGCCAGCGCGCTGCGCCCC-3' 5'-GGGGCGCAGCGCGCTGGCCAGGCTGTAGGT-3' Vimentin S41A 5'-TACAGCCTGGGCGCCGCGCTGCGCCCCAGC-3' 5'-GCTGGGGCGCAGCGCGGCGCCCAGGCTGTA-3' Vimentin L43A 5'-CTGGGCAGCGCGGCGCGCCCCAGCACCAGC-3' 5'-GCTGGTGCTGGGGCGCGCCGCGCTGCCCAG-3' Vimentin R44A 5'-GGCAGCGCGCTGGCCCCCAGCACCAGCCGC-3' 5'-GCGGCTGGTGCTGGGGGCCAGCGCGCTGCC-3' Vimentin P45A 5'-AGCGCGCTGCGCGCCAGCACCAGCCGCAGC-3' 5'-GCTGCGGCTGGTGCTGGCGCGCAGCGCGCT-3' Vimentin S46A 5'-GCGCTGCGCCCCGCCACCAGCCGCAGCCTC-3' 5'-GAGGCTGCGGCTGGTGGCGGGGCGCAGCGC-3' Desmin Y4A 5'-AGCCAGTCCGCTTCAAGCAACCAGAGGGCT-3' 5'-AGCCCTCTGGTTGCTTGAAGCGGACTGGCT-3' Desmin S5A 5'-AGCCAGTCCTATGCAAGCAACCAGAGGGCT-3' 5'-AGCCCTCTGGTTGCTTGCATAGGACTGGCT-3' Desmin S6A 5'-CAGTCCTATTCAGCCAACCAGAGGGCTTCC-3' 5'-GGAAGCCCTCTGGTTGGCTGAATAGGACTG-3'

Desmin N7A 5'-TCCTATTCAAGCGCCCAGAGGGCTTCCTCC-3' 5'-GGAGGAAGCCCTCTGGGCGCTTGAATAGGA-3' Desmin Q8A 5'-TATTCAAGCAACGCGAGGGCTTCCTCCTAC-3' 5'-GTAGGAGGAAGCCCTCGCGTTGCTTGAATA-3' Desmin R9A 5'-TCAAGCAACCAGGCGGCTTCCTCCTACCGC-3' 5'-GCGGTAGGAGGAAGCCGCCTGGTTGCTTGA-3' Desmin S11A 5'-AACCAGAGGGCTGCCTCCTACCGCCGCACT-3' 5'-AGTGCGGCGGTAGGAGGCAGCCCTCTGGTT-3' Myosin II T7A 5'-AAAAGAGCAAAGGCCAAGACCACCAAAAAG-3' 5'-CTTTTTGGTGGTCTTGGCCTTTGCTCTTTT-3' Myosin II K8A 5'-AGAGCAAAGACCGCGACCACCAAAAAGCGC-3' 5'-GCGCTTTTTGGTGGTCGCGGTCTTTGCTCT-3' Myosin II T9A 5'-GCAAAGACCAAGGCCACCAAAAAGCGCCCT-3' 5'-AGGGCGCTTTTTGGTGGCCTTGGTCTTTGC-3' Myosin II T10A 5'-AAGACCAAGACCGCCAAAAAGCGCCCTCAA-3' 5'-TTGAGGGCGCTTTTTGGCGGTCTTGGTCTT-3' Myosin II K11A 5'-ACCAAGACCACCGCAAAGCGCCCTCAACGC-3' 5'-GCGTTGAGGGCGCTTTGCGGTGGTCTTGGT-3' Myosin II K12A 5'-AAGACCACCAAAGCGCGCCCTCAACGCGCA-3' 5'-TGCGCGTTGAGGGCGCGCTTTGGTGGTCTT-3' Myosin II R13A 5'-ACCACCAAAAAGGCCCCTCAACGCGCAACA-3' 5'-TGTTGCGCGTTGAGGGGCCTTTTTGGTGGT-3'

Myosin II P14A 5'-ACCAAAAAGCGCGCTCAACGCGCAACATCC-3' 5'-GGATGTTGCGCGTTGAGCGCGCTTTTTGGT-3' GFAP I6A 5'-GAGAGGAGACGCGCCACCTCCGCTGCTCGC-3' 5'-GCGAGCAGCGGAGGTGGCGCGTCTCCTCTC-3' GFAP T7A 5'-AGGAGACGCATCGCCTCCGCTGCTCGCCGC-3' 5'-GCGGCGAGCAGCGGAGGCGATGCGTCTCCT-3' GFAP S8A 5'-AGACGCATCACCGCCGCTGCTCGCCGCTCC-3' 5'-GGAGCGGCGAGCAGCGGCGGTGATGCGTCT-3' GFAP R11A 5'-ACCTCCGCTGCTGCCCGCTCCTACGTCTCC-3' 5'-GGAGACGTAGGAGCGGGCAGCAGCGGAGGT-3' GFAP R12A 5'-TCCGCTGCTCGCGCCTCCTACGTCTCCTCA-3' 5'-TGAGGAGACGTAGGAGGCGCGAGCAGCGGA-3' GFAP S13A 5'-GCTGCTCGCCGCGCCTACGTCTCCTCAGGG-3' 5'-CCCTGAGGAGACGTAGGCGCGGCGAGCAGC-3' Emi2 P639A 5'-AAAGAAGCACTTGCTGGAAGTGCCCAAAGC-3' 5'-GCTTTGGGCACTTCCAGCAAGTGCTTCTTT-3' Emi2 G640A 5'-GAAGCACTTCCTGCAAGTGCCCAAAGCAAA-3' 5'-TTTGCTTTGGGCACTTGCAGGAAGTGCTTC-3' Emi2 S641A 5'-GCACTTCCTGGAGCTGCCCAAAGCAAAAGG-3' 5'-CCTTTTGCTTTGGGCAGCTCCAGGAAGTGC-3' Emi2 Q643A 5'-CCTGGAAGTGCCGCAAGCAAAAGGAATTTG-3' 5'-CAAATTCCTTTTGCTTGCGGCACTTCCAGG-3'

Emi2 S644A 5'-GGAAGTGCCCAAGCCAAAAGGAATTTGAAG-3' 5'-CTTCAAATTCCTTTTGGCTTGGGCACTTCC-3' Emi2 K645A 5'-AGTGCCCAAAGCGCAAGGAATTTGAAGAGA-3' 5'-TCTCTTCAAATTCCTTGCGCTTTGGGCACT-3' Emi2 R646A 5'-GCCCAAAGCAAAGCGAATTTGAAGAGACTT-3' 5'-AAGTCTCTTCAAATTCGCTTTGCTTTGGGC-3' Ikaros T140A 5'-AAAAGAAGCCACGCTGGAGAACGGCCCTTC-3' 5'-GAAGGGCCGTTCTCCAGCGTGGCTTCTTTT-3' Ikaros R143A 5'-CACACTGGAGAAGCGCCCTTCCAGTGCAAT-3' 5'-ATTGCACTGGAAGGGCGCTTCTCCAGTGTG-3' Ikaros S168A 5'-ATCAAGCTGCATGCCGGGGAGAAGCCCTTC-3' 5'-GAAGGGCTTCTCCCCGGCATGCAGCTTGAT-3' Ikaros K171A 5'-CATTCCGGGGAGGCGCCCTTCAAATGCCAC-3' 5'-GTGGCATTTGAAGGGCGCCTCCCCGGAATG-3' Ikaros T194A 5'-GGCCACCTGAGGGCGCACTCCGTTGGTAAA-3' 5'-TTTACCAACGGAGTGCGCCCTCAGGTGGCC-3' Ikaros H195A 5'-CACCTGAGGACGGCCTCCGTTGGTAAACCT-3' 5'-AGGTTTACCAACGGAGGCCGTCCTCAGGTG-3' Ikaros S196A 5'-CTGAGGACGCACGCCGTTGGTAAACCTCAC-3' 5'-GTGAGGTTTACCAACGGCGTGCGTCCTCAG-3' Ikaros V197A 5'-AGGACGCACTCCGCTGGTAAACCTCACAAA-3' 5'-TTTGTGAGGTTTACCAGCGGAGTGCGTCCT-3'

Ikaros G198A 5'-ACGCACTCCGTTGCTAAACCTCACAAATGT-3' 5'-ACATTTGTGAGGTTTAGCAACGGAGTGCGT-3' Ikaros K199A 5'-CACTCCGTTGGTGCACCTCACAAATGTGGA-3' 5'-TCCACATTTGTGAGGTGCACCAACGGAGTG-3' Ikaros P200A 5'-TCCGTTGGTAAAGCTCACAAATGTGGATAT-3' 5'-ATATCCACATTTGTGAGCTTTACCAACGGA-3' Ikaros H201A 5'-GTTGGTAAACCTGCCAAATGTGGATATTGT-3' 5'-ACAATATCCACATTTGGCAGGTTTACCAAC-3' Sp1 T643A 5'-TTGCGCTGGCATGCAGGCGAGAGGCCATTT-3' 5'-AAATGGCCTCTCGCCTGCATGCCAGCGCAA-3' Sp1 R646A 5'-CATACAGGCGAGGCGCCATTTATGTGTACC-3' 5'-GGTACACATAAATGGCGCCTCGCCTGTATG-3' Sp1 T673A 5'-AAACGTACACACGCAGGTGAGAAGAAATTT-3' 5'-AAATTTCTTCTCACCTGCGTGTGTACGTTT-3' Sp1 K676A 5'-CACACAGGTGAGGCGAAATTTGCCTGCCCT-3' 5'-AGGGCAGGCAAATTTCGCCTCACCTGTGTG-3' YY1 T348A 5'-CAACTGGTTCATGCTGGAGAGAAGCCCTTT-3' 5'-AAAGGGCTTCTCTCCAGCATGAACCAGTTG-3' YY1 K1A 5'-CATACTGGAGAGGCGCCCTTTCAGTGCACG-3' 5'-CGTGCACTGAAAGGGCGCCTCTCCAGTATG-3' YY1 T378A 5'-GTGCGAATCCATGCCGGAGACAGGCCCTAT-3' 5'-ATAGGGCCTGTCTCCGGCATGGATTCGCAC-3'

YY1 R381A 5'-CATACCGGAGACGCGCCCTATGTGTGCCCC-3' 5'-GGGGCACACATAGGGCGCGTCTCCGGTATG-3' Ect2 T342A 5'-CTTTCTCTAAATGCCCCTAACAGCAATCGC-3' 5'-GCGATTGCTGTTAGGGGCATTTAGAGAAAG-3' Ect2 S345A 5'-AATACCCCTAACGCCAATCGCAAACGACGT-3' 5'-ACGTCGTTTGCGATTGGCGTTAGGGGTATT-3' Ect2 T342A/S345A 5'-TCTCTAAATGCCCCTAACGCCAATCGCAAA-3' 5'-TTTGCGATTGGCGTTAGGGGCATTTAGAGA-3' Ect2 T342E 5'-CTTTCTCTAAATCAGCCTAACAGCAATCGC-3' 5'-GCGATTGCTGTTAGGCTGATTTAGAGAAAG-3' Ect2 S345D 5'-AATACCCCTAACGACAATCGCAAACGACGT-3' 5'-ACGTCGTTTGCGATTGTCGTTAGGGGTATT-3' Ect2 T342E/S345D 5'-TCTCTAAATGAGCCTAACGACAATCGCAAA-3' 5'-TTTGCGATTGTCGTTAGGCTCATTTAGAGA-3' Ect2 KRR/3A 5'-AACAGCAATCGCGCAGCAGCTCGTTTAAAAGAA-3' 5'-TTCTTTTAAACGAGCTGCTGCGCGATTGCTGTT-3' Ect2 RKRRR/5A 5'-CCTAACAGCAATGCCGCAGCAGCTGCTTTAAAAGAAACA-3' 5'-TGTTTCTTTTAAAGCAGCTGCTGCGGCATTGCTGTTAGG-3' Cullin 4B T31A 5'-AATACCAGCACCGCTCCGCCCACCTCTGCC-3' 5'-GGCAGAGGTGGGCGGAGCGGTGCTGGTATT-3' Cullin 4B SA 5'-ACTCCGCCCACCGCTGCCAAGAAGAGAAAG-3' 5'-CTTTCTCTTCTTGGCAGCGGTGGGCGGAGT-3'

Cullin 4B T31A/SA 5'-AGCACCGCTCCGCCCACCGCTGCCAAGAAG-3' 5'-CTTCTTGGCAGCGGTGGGCGGAGCGGTGCT-3' Cullin 4B KR/AA 5'-ACCTCTGCCAAGGCGGCAAAGTTAAACAGC-3' 5'-GCTGTTTAACTTTGCCGCCTTGGCAGAGGT-3'

Supplementary Table S2. Antibodies. Antibody Cat. Number Species Dilution Source Anti-Flag M2 F1804 mouse 1 : 1000 Sigma Anti-HpTGEKP motif ABE319 rabbit 1 : 500 Millipore Anti-Importnin β ab36775 rabbit 1 : 1000 abcam Anti-Xenopus cyclin B1 - sheep 1 : 1000 (serum) J. Maller

Supplementary Reference 1. Dephoure, N. et al. A quantitative atlas of mitotic phosphorylation. Proc. Natl Acad. Sci. USA 105, 10762-10767 (2008).

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