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| | Name | Initial Conc. (uM) | Volume (fL) | Buffered | | 1 | AA | 6.12 | 1000 | No | | | Arachidonic Acid. This messenger diffuses through membranes as well as cytosolically, has been suggested as a possible retrograde messenger at synapses. | | 2 | Ca | 0.08 | 1000 | Yes | | | This calcium pool is treated as being buffered to a steady 0.08 uM, which is the resting level. | | 3 | DAG | 11.661 | 1000 | Yes | | | Baseline in model is 11.661 uM. DAG is pretty nasty to estimate. In this model we just hold it fixed at this baseline level. Data sources are many and varied and sometimes difficult to reconcile. Welsh CJ and Cabot MC (1987) J Cell Biochem. 35(3):231-245. DAG degradation Bocckino SB, Blackmore PF, Exton JH. (1985) J Biol Chem. 260(26):14201-14207. Hepatocytes stimulated with vasopressin: 190 uM. Bocckino SB, Blackmore PF, Wilson PB, Exton JH. (1987) J Biol Chem. 262(31):15309-15. DAG rises from 70 to 200 ng/mg wet weight, approx 150 to 450 uM. Prescott SM and Majerus PW. (1983) J Biol Chem. 258(2):764-769. Platelets: 6 uM. Also see Rittenhouse-Simmons S. (1979) J Clin Invest. 63(4):580-587. Sano K, Takai Y, Yamanishi J, Nishizuka Y. (1983) J Biol Chem. 258(3):2010-2013. report a nearly 10 fold rise. Habenicht AJ, Glomset JA, King WC, Nist C, Mitchell CD, Ross R. (1981) J Biol Chem. 256(23):12329-35. 3T3 cells with PDGF stim: 27 uM. Cornell R and Vance DE. (1987) Biochim Biophys Acta. 1987 May 13;919(1):26-36. 10x rise from 10 to 100 uM.
Summary: I see much lower rises in my PLC models, but the baseline could be anywhere from 5 to 100 uM. I have chosen about 11 uM based on the stimulus -response characteristics from Schaechter JD and Benowitz LI. (1993) J Neurosci. 13(10):4361-4371 and Shinomura T, Asaoka Y, Oka M, Yoshida K, Nishizuka Y. (1991) Proc Natl Acad Sci U S A. 88(12):5149-53. | | 4 | MAPK* | 0 | 1000 | No | | | This molecule is phosphorylated on both the tyr and thr residues and is active: Seger R, Ahn NG, Posada J, Munar ES, Jensen AM, Cooper JA, Cobb MH, Krebs EG. (1992) J Biol Chem. 267(20):14373-81. The rate constants are from two sources - combine Sanghera JS, Paddon HB, Bader SA, Pelech SL. (1990) J Biol Chem. 265(1):52-57 with Nemenoff RA, Winitz S, Qian NX, Van Putten V, Johnson GL, Heasley LE. (1993) J Biol Chem. 268(3):1960-1964 to get k3 = 10, k2 = 40, k1 = 3.25e-6
| | 5 | MKP-1 | 0.0004 | 1000 | No | | | MKP-1 dephosphorylates and inactivates MAPK in vivo: Sun H, Charles CH, Lau LF, Tonks NK. (1993) Cell 75(3):487-493. See Charles CH, Sun H, Lau LF, Tonks NK. (1993) Proc Natl Acad Sci U S A. 90(11):5292-5296 and Charles CH, Abler AS, Lau LF. (1992) Oncogene 7(1):187-190 for half-life of MKP1/3CH is 40 min. 80% deph of MAPK in 20 min The protein is 40 KDa. 22 Apr 2001: CoInit =0.4nM but this is really an emergent property of the rate of induction of the phosphatase at steady-state in balance with the degradation rate.
| | 6 | MKP-2 | 0.002 | 1000 | No | | | MKP2 is modeled to act as a relatively steady, unregulated phosphatase for controlling MAPK activity. From Brondello JM, Brunet A, Pouyssegur J, McKenzie FR (1997) J Biol Chem. 272(2):1368-1376, the blockage of MKP-1 induction increases MAPK activity by no more than 2x. So this phosphatase will play the steady role and the fully stimulated MKP-1 can come up to the level of this steady level. From Chu Y, Solski PA, Khosravi-Far R, Der CJ, Kelly K (1996) J Biol Chem. 271(11):6497-6501 it looks like both MKP-1 and MKP-2 have similar activities in dephosphorylating ERK2. So I use the same enzymatic rates for both. | | 7 | PKC-active | 0.02 | 1000 | No | | | This is the total active PKC. It is the sum of the respective activities of PKC-basal* PKC-Ca-memb* PKC-DAG-memb* PKC-Ca-AA* PKC-DAG-AA* PKC-AA* I treat PKC here in a two-state manner: Either it is in an active state (any one of the above list) or it is inactive. No matter what combination of stimuli activate the PKC, I treat it as having the same activity. The scaling comes in through the relative amounts of PKC which bind to the respecive stimuli. The justification for this is the mode of action of PKC, which like most Ser/Thr kinases has a kinase domain normally bound to and blocked by a regulatory domain. I assume that all the activators simply free up the kinase domain. A more general model would incorporate a different enzyme activity for each combination of activating inputs, as well as for each substrate. The current model seems to be a decent and much simpler approximation for the available data. One caveat of this way of representing PKC is that the summation procedure assumes that PKC does not saturate with its substrates. If this assumption fails, then the contributing PKC complexes would experience changes in availability which would affect their balance. Given the relatively low percentage of PKC usually activated, and its high throughput as an enzyme, this is a safe assumption under physiological conditions. | | 8 | PPhosphatase2A | 0.224 | 1000 | No | | | CoInit values span a range depending on the source Pato MD, Sutherland C, Winder SJ, Walsh MP. (1993) Biochem J. 293 ( Pt 1):35-41; and Cohen P, Alemany S, Hemmings BA, Resink TJ, Stralfors P, Tung HY. (1988) Methods Enzymol. 159:390-408 estimate 80 nM from muscle. Zolnierowicz S, Csortos C, Bondor J, Verin A, Mumby MC, DePaoli-Roach AA. (1994) Biochemistry 33(39):11858-11867 report levels of 0.4 uM again from muscle, but expression is also strong in brain. Our estimate of 0.224 is between these two.
There are many substrates for PP2A in this model, so I put the enzyme rate calculations here: Takai A and Mieskes G (1991) Biochem J. 275 ( Pt 1):233-9 have mol wt 36 KDa. They report Vmax of 119 umol/min/mg i.e. 125/sec for k3 for pNPP substrate, Km of 16 mM. This is obviously unreasonable for protein substrates. For chicken gizzard myosin light chan, we have Vmax = 13 umol/min/mg or about k3 = 14/sec. Pato MD, Sutherland C, Winder SJ, Walsh MP. (1993) Biochem J. 293 ( Pt 1):35-41 report caldesmon: Km = 2.2 uM, Vmax = 0.24 umol/min/mg. They do not think caldesmon is a good substrate. Calponin: Km = 14.3, Vmax = 5. Our values approximate these. | | 9 | Shc*.Sos.Grb2 | 0 | 1000 | No | | | This three-way complex is one of the main GEFs for activating Ras. | | 10 | temp-PIP2 | 2.5 | 1000 | Yes | | | This is a steady PIP2 input to PLA2. The sensitivity of PLA2 to PIP2 discussed below does not match with the reported free levels which are used by the phosphlipase Cs. My understanding is that there may be different pools of PIP2 available for stimulating PLA2 as opposed to being substrates for PLCs. For that reason I have given this PIP2 pool a separate identity. As it is a steady input this is not a problem in this model. Majerus PW, Neufeld EJ, Wilson DB. (1984) Cell 37(3):701-703 report a brain concentration of 0.1 - 0.2 mole %. Majerus PW, Connolly TM, Deckmyn H, Ross TS, Bross TE, Ishii H, Bansal VS, Wilson DB. (1986) Science 234(4783):1519-1526 report a huge range of concentrations: from 1 to 10% of PI content, which is in turn 2-8% of cell lipid. This gives 2e-4 to 8e-3 of cell lipid. In concentrations in total volume of cell (a somewhat strange number given the compartmental considerations) this comes to anywhere from 4 uM to 200 uM. PLA2 is stim 7x by PIP2. [Leslie CC and Channon JY (1990) Biochim Biophys Acta. 1045(3):261-270] Leslie and Channon say PIP2 is present at 0.1 - 0.2mol% range in membs, so I'll use a value at the lower end of the scale for basal PIP2. | | 11 | tot_MAPK | 0 | 1000 | No | | | Total available active MAPK. This sums the levels of the cytosolic and nuclear localized forms. | | 12 | tot_MKP1 | 0 | 1000 | No | | | Total available active MKP-1. This sums the levels of the non-phosphorylated, singly phosphorylated and doubly phosphorylated forms. It is used primarily for graphing the total MKP-1 level. |
Summed Molecule List | | Target | Inputs | | 1 |
PKC-active | PKC-DAG-AA*
PKC-Ca-memb*
PKC-Ca-AA*
PKC-DAG-memb*
PKC-basal*
PKC-AA*
| This is the total active PKC. It is the sum of the respective activities of PKC-basal* PKC-Ca-memb* PKC-DAG-memb* PKC-Ca-AA* PKC-DAG-AA* PKC-AA* I treat PKC here in a two-state manner: Either it is in an active state (any one of the above list) or it is inactive. No matter what combination of stimuli activate the PKC, I treat it as having the same activity. The scaling comes in through the relative amounts of PKC which bind to the respecive stimuli. The justification for this is the mode of action of PKC, which like most Ser/Thr kinases has a kinase domain normally bound to and blocked by a regulatory domain. I assume that all the activators simply free up the kinase domain. A more general model would incorporate a different enzyme activity for each combination of activating inputs, as well as for each substrate. The current model seems to be a decent and much simpler approximation for the available data. One caveat of this way of representing PKC is that the summation procedure assumes that PKC does not saturate with its substrates. If this assumption fails, then the contributing PKC complexes would experience changes in availability which would affect their balance. Given the relatively low percentage of PKC usually activated, and its high throughput as an enzyme, this is a safe assumption under physiological conditions. | | 2 |
tot_MAPK | MAPK*
nuc_MAPK*
| Total available active MAPK. This sums the levels of the cytosolic and nuclear localized forms. | | 3 |
tot_MKP1 | MKP-1
MKP-1-ser359*
MKP-1**
| Total available active MKP-1. This sums the levels of the non-phosphorylated, singly phosphorylated and doubly phosphorylated forms. It is used primarily for graphing the total MKP-1 level. |
Pathway Details Molecule List Enzyme List Reaction List
| Database compilation and code copyright (C) 2022, Upinder S. Bhalla and NCBS/TIFR
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