| | Name | Initial Conc. (uM) | Volume (fL) | Buffered |
| 1 | craf-1 | 0.5 | 1.5 | No |
| | Couldn't find any ref to the actual conc of craf-1 but I should try Strom et al Oncogene 5 pp 345 In line with the other kinases in the cascade, I estimate the conc to be 0.2 uM. To init we use 0.15, which is close to equil 16 May 2003: Changing to synaptic levels. Increasing 2.5 fold to 0.5 uM. See Mihaly et al 1991 Brain Res 547(2):309-14 and Morice et al 1999 Eur J Neurosci 11(6):1995-2006 |
| 2 | craf-1* | 0 | 1.5 | No |
| 3 | craf-1** | 0 | 1.5 | No |
| | Negative feedback by MAPK* by hyperphosphorylating craf-1* gives rise to this pool. Ueki et al JBC 269(22):15756-15761, 1994 |
| 4 | MAPK | 3.5999 | 1.5 | No |
| | conc is from Sanghera et al JBC 265 pp 52 (1990) A second calculation gives 3.1 uM, from same paper. They est MAPK is 1e-4x total protein, and protein is 15% of cell wt, so MAPK is 1.5e-5g/ml = 0.36uM. which is closer to our first estimate. Lets use this. Updated 16 May 2003. Ortiz et al 1995 J Neurosci 15(2):1285-1297 provide estimates of ERK2 levels in hippocampus: 1009 ng/mg. This comes to about 3.6uM, which may still be an underestimate of synaptic levels. |
| 5 | MAPK-tyr | 0 | 1.5 | No |
| | Haystead et al FEBS Lett. 306(1) pp 17-22 show that phosphorylation is strictly sequential, first tyr185 then thr183. |
| 6 | MAPKK | 0.5 | 1.5 | No |
| | Conc is from Seger et al JBC 267:20 pp14373 (1992) mwt is 45/46 Kd We assume that phosphorylation on both ser and thr is needed for activiation. See Kyriakis et al Nature 358 417 1992 Init conc of total is 0.18 Ortiz et al 1995 J Neurosci 15(2):1285-1297 suggest that levels are higher in hippocampus than other brain regions, and further elevated in synapses. Estimate 3x higher levels than before, at 0.5 uM. Similar results from Schipper et al 1999 Neuroscience 93(2):585-595 but again lacking in quantitation. |
| 7 | MAPKK* | 0 | 1.5 | No |
| | MAPKK phosphorylates MAPK on both the tyr and thr residues, first tyr then thr. Refs: Seger et al JBC267:20 pp 14373 1992 The MAPKK itself is phosphorylated on ser as well as thr residues. Let us assume that the ser goes first, and that the sequential phosphorylation is needed. See Kyriakis et al Nature 358 417-421 1992 |
| 8 | MAPKK-ser | 0 | 1.5 | No |
| | Intermediately phophorylated, assumed inactive, form of MAPKK |
| 9 | Raf*-GTP-Ras | 0 | 1.5 | No |
| 10 | Raf-GTP-Ras | 0 | 1.5 | No |
