In vitro phosphorylation of potential cyclin E–Cdk2 substrates by different cyclin–Cdk complexes
| Potential cyclin E–Cdk2 substrate . | . | . | Phosphorylation sites and cyclin binding motifs in the RZPD clone . | . | . | Phosphorylation of the RZPD clone by kinasesa . | . | . | . | . | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Gene . | ORF of RZPD clone . | Name/biological process . | min: S/TP . | cons: S/TPXK/R . | RXL . | D1–K4 . | D3–K4 . | E–K2 . | A–K2 . | B–K1 . | ||||||||
| NUMA1b | 245 aa C-term | Nuclear mitotic apparatus protein 1/ spindle apparatus organization | 7 | 1 | 0 | − | ± | + | + | − | ||||||||
| LOC201191 | 231 aa C-term | Hypothetical protein LOC201191/unknown | 6 | 1 | 2 | − | ± | + | + | − | ||||||||
| PIP5K1C | 200 aa C-term | Phosphatidylinositol-4-phosphate 5 kinase, type I γ/phosphatidylinositol signaling | 7 | 2 | 1 | ± | + | + | + | ± | ||||||||
| CCNL2 | 136 aa C-term | cyclin L2 / RNA processing | 7 | 3 | 2 | − | − | ± | + | ± | ||||||||
| RASL11B | 118 aa C-term | RAS-like, family 11, member B/signaling? | 2 | 1 | 0 | − | + | + | + | − | ||||||||
| SIRT2 | full-lengthc | Sirtuin (silent mating type information regulation 2 homologue) 2 (S. cerevisiae)/ NAD+-dependent histone/protein deacetylase | 3 | 1 | 1 | − | − | + | + | ± | ||||||||
| FLJ13111 | full-length | Hypothetical protein FLJ13111/unknown | 4 | 2 | 4 | − | ± | + | + | ± | ||||||||
| SUPT5Hb | 120 aa C-term | Suppressor of Ty 5 homologue (S. cerevisiae)/transcription | 1 | 1 | 1 | − | − | ± | − | − | ||||||||
| STMN2b | full-length | Stathmin-like 2/microtubule destabilization in neuronal growth | 2 | 1 | 1 | − | + | + | + | − | ||||||||
| LOC146909 | 114 aa C-term | Hypothetical protein LOC146909/unknown | 1 | 0 | 1 | − | + | + | + | − | ||||||||
| EEF1G | 211 aa C-term | Eukaryotic translation elongation factor 1γ/translation | 2 | 1 | 1 | − | ± | + | + | ± | ||||||||
| TRIT1 | 99 aa C-term | tRNA isopentenyltransferase 1/ RNA modification | 3 | 1 | 1 | − | + | + | + | ± | ||||||||
| LOC388799 | 89 aa C-term | Hypothetical protein LOC388799/unknown | 2 | 1 | 0 | − | ± | + | + | ± | ||||||||
| STUB1 | full-length | STIP1 homology and U-box–containing protein 1/protein turnover | 4 | 1 | 3 | − | + | + | + | ± | ||||||||
| SRRM2 | 98 aa central | Serine/arginine repetitive matrix 2/ RNA processing | 3 | 0 | 0 | − | − | + | + | + | ||||||||
| TALDO1 | full-length | Transaldolase 1/metabolism | 3 | 1 | 6 | − | + | + | + | − | ||||||||
| SFRS1 | 243 aa C-term | Splicing factor, arginine/serine-rich 1 (splicing factor 2, alternate splicing factor)/RNA processing | 4 | 1 | 1 | − | + | + | + | − | ||||||||
| SNAPAP | full-length | SNAP-associated protein/exocytosis of synaptic vesicles | 2 | 1 | 2 | + | ± | + | ||||||||||
| LOC339287 | 90 aa central | Hypothetical protein LOC339287/unknown | 5 | 4 | 1 | + | + | + | + | + | ||||||||
| FLJ12949 | 248 aa C-term | Hypothetical protein FLJ12949/unknown | 4 | 1 | 3 | − | + | + | − | − | ||||||||
| MAP2 | 376 aa C-term | MAP2/microtubule assembly in neurogenesis | 16 | 3 | 1 | − | + | + | + | − | ||||||||
| RPL14 | 211 aa C-term | Ribosomal protein L14/translation | 1 | 1 | 1 | − | ± | − | − | − | ||||||||
| FNBP3 | 245 aa C-term | Formin binding protein 3/splicing? | 2 | 1 | 1 | − | ± | ± | ± | − | ||||||||
| PRC1d | full-length | Protein regulating cytokinesis 1/ cytokinesis | 5 | 2 | 2 | − | + | + | + | ± | ||||||||
| FLJ13305 | 162 aa central | Hypothetical protein FLJ13305/unknown | 4 | 0 | 3 | − | + | + | ± | − | ||||||||
| ING5 | full-lengthc | Inhibitor of growth family, member 5/ p53 pathway, replication | 2 | 1 | 3 | − | − | + | + | − | ||||||||
| Potential cyclin E–Cdk2 substrate . | . | . | Phosphorylation sites and cyclin binding motifs in the RZPD clone . | . | . | Phosphorylation of the RZPD clone by kinasesa . | . | . | . | . | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Gene . | ORF of RZPD clone . | Name/biological process . | min: S/TP . | cons: S/TPXK/R . | RXL . | D1–K4 . | D3–K4 . | E–K2 . | A–K2 . | B–K1 . | ||||||||
| NUMA1b | 245 aa C-term | Nuclear mitotic apparatus protein 1/ spindle apparatus organization | 7 | 1 | 0 | − | ± | + | + | − | ||||||||
| LOC201191 | 231 aa C-term | Hypothetical protein LOC201191/unknown | 6 | 1 | 2 | − | ± | + | + | − | ||||||||
| PIP5K1C | 200 aa C-term | Phosphatidylinositol-4-phosphate 5 kinase, type I γ/phosphatidylinositol signaling | 7 | 2 | 1 | ± | + | + | + | ± | ||||||||
| CCNL2 | 136 aa C-term | cyclin L2 / RNA processing | 7 | 3 | 2 | − | − | ± | + | ± | ||||||||
| RASL11B | 118 aa C-term | RAS-like, family 11, member B/signaling? | 2 | 1 | 0 | − | + | + | + | − | ||||||||
| SIRT2 | full-lengthc | Sirtuin (silent mating type information regulation 2 homologue) 2 (S. cerevisiae)/ NAD+-dependent histone/protein deacetylase | 3 | 1 | 1 | − | − | + | + | ± | ||||||||
| FLJ13111 | full-length | Hypothetical protein FLJ13111/unknown | 4 | 2 | 4 | − | ± | + | + | ± | ||||||||
| SUPT5Hb | 120 aa C-term | Suppressor of Ty 5 homologue (S. cerevisiae)/transcription | 1 | 1 | 1 | − | − | ± | − | − | ||||||||
| STMN2b | full-length | Stathmin-like 2/microtubule destabilization in neuronal growth | 2 | 1 | 1 | − | + | + | + | − | ||||||||
| LOC146909 | 114 aa C-term | Hypothetical protein LOC146909/unknown | 1 | 0 | 1 | − | + | + | + | − | ||||||||
| EEF1G | 211 aa C-term | Eukaryotic translation elongation factor 1γ/translation | 2 | 1 | 1 | − | ± | + | + | ± | ||||||||
| TRIT1 | 99 aa C-term | tRNA isopentenyltransferase 1/ RNA modification | 3 | 1 | 1 | − | + | + | + | ± | ||||||||
| LOC388799 | 89 aa C-term | Hypothetical protein LOC388799/unknown | 2 | 1 | 0 | − | ± | + | + | ± | ||||||||
| STUB1 | full-length | STIP1 homology and U-box–containing protein 1/protein turnover | 4 | 1 | 3 | − | + | + | + | ± | ||||||||
| SRRM2 | 98 aa central | Serine/arginine repetitive matrix 2/ RNA processing | 3 | 0 | 0 | − | − | + | + | + | ||||||||
| TALDO1 | full-length | Transaldolase 1/metabolism | 3 | 1 | 6 | − | + | + | + | − | ||||||||
| SFRS1 | 243 aa C-term | Splicing factor, arginine/serine-rich 1 (splicing factor 2, alternate splicing factor)/RNA processing | 4 | 1 | 1 | − | + | + | + | − | ||||||||
| SNAPAP | full-length | SNAP-associated protein/exocytosis of synaptic vesicles | 2 | 1 | 2 | + | ± | + | ||||||||||
| LOC339287 | 90 aa central | Hypothetical protein LOC339287/unknown | 5 | 4 | 1 | + | + | + | + | + | ||||||||
| FLJ12949 | 248 aa C-term | Hypothetical protein FLJ12949/unknown | 4 | 1 | 3 | − | + | + | − | − | ||||||||
| MAP2 | 376 aa C-term | MAP2/microtubule assembly in neurogenesis | 16 | 3 | 1 | − | + | + | + | − | ||||||||
| RPL14 | 211 aa C-term | Ribosomal protein L14/translation | 1 | 1 | 1 | − | ± | − | − | − | ||||||||
| FNBP3 | 245 aa C-term | Formin binding protein 3/splicing? | 2 | 1 | 1 | − | ± | ± | ± | − | ||||||||
| PRC1d | full-length | Protein regulating cytokinesis 1/ cytokinesis | 5 | 2 | 2 | − | + | + | + | ± | ||||||||
| FLJ13305 | 162 aa central | Hypothetical protein FLJ13305/unknown | 4 | 0 | 3 | − | + | + | ± | − | ||||||||
| ING5 | full-lengthc | Inhibitor of growth family, member 5/ p53 pathway, replication | 2 | 1 | 3 | − | − | + | + | − | ||||||||
The kinases used are abbreviated (e.g., D1–K4 for cyclin D1–Cdk4). The substrates are phosphorylated strongly (+), weakly (±), or not at all (−) by the respective kinase.
Previously reported Cdk substrate (Stachora et al., 1997; Gavet et al., 1998; Sun and Schatten, 2006).
For ING5 and SIRT2, GST-tagged full-length proteins were used in the kinase assays.
Previously reported cyclin E–Cdk2 substrate (Jiang et al., 1998).