Table 2.
In vitro phosphorylation of synthetic peptides modeled after SARS-CoV-2 nucleocapsid protein phosphorylation sites by recombinant protein kinase C-alpha and glycogen synthase kinase-beta using the ADP-Glo method
| Peptide | Peptide Sequence | PKCa | GSK3b | PKCa+GSK3b |
|---|---|---|---|---|
| SCV2-N [177-190] - pS188 | RGGSQASSRSS(pS)RS | 3861 ± 71 | 24,585 ± 645 | NT |
| SCV2-N [177-190] | RGGSQASSRSSSRS | 13,714 ± 502 | 2161 ± 63 | 3572 ± 22 |
| SCV2-N [180-193] - pS190 | SQASSRSSSR(pS)RNS | 4161 ± 66 | 2957 ± 3 | NT |
| SCV2-N [180-193] | SQASSRSSSRSRNS | 16,366 ± 261 | 2407 ± 23 | 3836 ± 41 |
| SCV2-N [187-200] - pT198 | SSRSRNSSRNS(pT)PG | 7032 ± 319 | 23,031 ± 486 | NT |
| SCV2-N [187-200] | SSRSRNSSRNSTPG | 12,687 ± 486 | 2573 ± 49 | 3692 ± 61 |
| SCV2-N [195-209] - pS206 | RNSTPGSSRGT(pS)PAR | 1531 ± 47 | 24,082 ± 381 | NT |
| SCV2-N [195-209] | RNSTPGSSRGTSPAR | 3301 ± 114 | 3291 ± 57 | 4262 ± 27 |
| Nucleocapsid proteina | Full length | 1032 ± 9 | 2052 ± 90 | 2754 ± 66 |
| No peptide substrate | Autophosphorylation | 1282 ± 51 | 1995 ± 133 | 2897 ± 38 |
Predicted GSK3 phosphorylation sites are bolded and underlined. Results are presented as Mean ± Standard deviation, n = 3
NT not tested
aRelative phosphorylation is shown in comparison to the signal with the nucleocapsid protein incubated in the absence of added substrate with ATP alone (set to 100)