Table 1. Phosphorylation by protein kinase CK1 of peptides derived from β-catenin, NF-AT4, and APC and canonical peptides from β-casein and inhibitor-2 of protein phosphatase 1.
|
Rat liver CK1
|
CK1 α zebrafish
|
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|---|---|---|---|---|---|---|---|
| Short name | Sequence | Km, μM | Vmax, cpm | Vmax/km | Km, μM | Vmax, cpm | Vmax/km |
| BC-WT or parent | RRRGATTTAPSLSGKGNPEDEDVDTNQVLYE | 714 | 43,478 | 61 | 1,260 | 44,604 | 35 |
| BC S45A | RRRGATTTAPALSGKGNPEDEDVDTNQVLYE | 715 | 3,644 | 5 | nd | nd | 0 |
| BC S45T | RRRGATTTAPTLSGKGNPEDEDVDTNQVLYE | 357 | 4,347 | 12 | 821 | 5,352 | 6 |
| BC pS45 | RRRGATTTAPpSLSGKGNPEDEDVDTNQVLYE | nd | nd | 0 | nd | nd | 0 |
| BC L46A | RRRGATTTAPSASGKGNPEDEDVDTNQVLYE | 988 | 4,166 | 4 | 2,159 | 1,784 | 1 |
| BC L46V | RRRGATTTAPSVSGKGNPEDEDVDTNQVLYE | 1,060 | 31,279 | 29 | 821 | 25,870 | 31 |
| BC L46I | RRRGATTTAPSISGKGNPEDEDVDTNQVLYE | 1,330 | 25,575 | 19 | 780 | 28,546 | 36 |
| BCL46(d-Leu) | RRRGATTTAPS(D-L)SGKGNPEDEDVDTNQVLYE | 1,050 | 2,857 | 3 | nd | nd | 0 |
| BCL46(±t-Bu-Gly) | RRRGATTTAPS(tBu-Gly)SGKGNPEDEDVDTNQVLYE | 800 | 93,940 | 117 | — | — | — |
| BC S47A | RRRGATTTAPSLAGKGNPEDEDVDTNQVLYE | 714 | 16,666 | 23 | 1,413 | 22,748 | 16 |
| BC S47N | RRRGATTTAPSLNGKGNPEDEDVDTNQVLYE | 1,330 | 10,869 | 8 | 585 | 8,028 | 14 |
| BCS47T | RRRGATTTAPSLTGKGNPEDEDVDTNQVLYE | 1,031 | 17,391 | 17 | 505 | 10,285 | 20 |
| BC E53A,E55A,D58A | RRRGATTTAPSLSGKGNPADADVATNQVLYE | 2,000 | 9,803 | 5 | 2,802 | 29,884 | 11 |
| BC acidic all A | RRRGATTTAPSLSGKGNPAAAAVATNQVLYE | 1,612 | 2,337 | 1.5 | — | — | — |
| BC E55K | RRRGATTTAPSLSGKGNPEDKDVDTNQVLYE | 2,850 | 21,739 | 8 | 2,956 | 25,478 | 9 |
| BC N-terminal (Δ52-65) | RRRGATTTAPSLSGKGN————- | 2,127 | 5,955 | 3 | 4,581 | 5,352 | 1 |
| BC C-terminal (Δ38-48) | RRR———-KGNPEDEDVDTNQVLYE | 1,282 | 988 | 1 | nd | nd | 0 |
| BC P44S | RRRGATTTASSLSGKGNPEDEDVDTNQVLYE | 1,200 | 15,202 | 13 | — | — | — |
| BC P52A | RRRGATTTAPSLSGKGNAEDEDVDTNQVLYE | 1,372 | 20,869 | 15 | 2,992 | 31,222 | 10 |
| BCΔ48-51 | RRRGATTTAPSLS—PEDEDVDTNQVLYE | 2,000 | 26,191 | 13 | 3,205 | 21,588 | 7 |
| BC 48-51 all A | RRRGATTTAPSLSAAAAPEDEDVDTNQVLYE | 833 | 6,793 | 8 | 1,544 | 4,461 | 3 |
| BC K49R | RRRGATTTAPSLSGRGNPEDEDVDTNQVLYE | 500 | 38,476 | 77 | — | — | — |
| β-casein (Ser-22) | ...pSpSpSEES22ITRI... | 44 | 72,608 | 1,650 | — | — | — |
| Inhibitor-2 peptide | RRKHAAIGDDDDAYSITA | 363 | 373,910 | 1,030 | — | — | — |
| APC (1275-1290) | RRRASSLSSLSSAEDEIGCN | 140 | 24,844 | 177 | — | — | — |
| APC L (1278, 1281)A | RRRASSASSASSAEDEIGCN | nd | nd | 0 | |||
| NF-AT4 S194A (183-202) | RRRAESLSHIYDDVDAELNEAAAR | 909 | 14,187 | 16 | — | — | — |
Peptides preparation is described in Materials and Methods. The parent or WT peptide of β-catenin (BC) contained the sequence of X. laevis β-catenin corresponding to residues 38-65. The NF-AT4 peptide contained the sequence of the human NF-AT4 protein from residues 183-202 with a sequence RRRA attached to its amino end. The APC peptides contain the sequence of the human APC protein from residues 1275 to 1290 or mutants with a sequence RRRA attached to its amino end. Peptides from β-casein and protein phosphatase 1 inhibitor-2 had been studied (21, 24). All other peptides contain three arginines in their amino terminus to facilitate the phosphocellulose paper assay. nd, nondetectable phosphorylation; —, assay not performed.