Multiple Decisive Phosphorylation Sites for the Negative Feedback Regulation of SOS1 via ERK*

Abstract

EGF-induced activation of ERK has been extensively studied by both experimental and theoretical approaches. Here, we used a simulation model based mostly on experimentally determined parameters to study the ERK-mediated negative feedback regulation of the Ras guanine nucleotide exchange factor, son of sevenless (SOS). Because SOS1 is phosphorylated at multiple serine residues upon stimulation, we evaluated the role of the multiplicity by building two simulation models, which we termed the decisive and cooperative phosphorylation models. The two models were constrained by the duration of Ras activation and basal phosphorylation level of SOS1. Possible solutions were found only in the decisive model wherein at least three, and probably more than four, phosphorylation sites decisively suppress the SOS activity. Thus, the combination of experimental approaches and the model analysis has suggested an unexpected role of multiple phosphorylations of SOS1 in the negative regulation.

Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

“…Most of the enzymatic reactions were described as first-order reactions for simplicity. The reactions that follow the first-order enzymatic kinetics in our model (see Among them, MEK dephosphorylation and ERK dephosphorylation were measured in our previous study (9,22). The other reactions were approximated as first-order reactions for simplicity.…”

“…The cDNAs of human RSK1 and human RSK3 were amplified by PCR. The cDNAs of human Shc1 (20), human Grb2 (21), mouse Sos1 (22), mouse p85, cow p110␣ (23), human HRasdCT-G12V (where dCT stands for deleted C terminus) (24), human BRaf, human BRaf-S364A (25), human CRaf, human CRaf-S259A (24), human RSK2 (26), human RSK1, and human RSK3 were subcloned into mEGFP or HaloTag vector to generate pCXN2-mEGFP-Shc1-stop, pCX4puro-HaloTag-Shc, pCAGGS-FLAGGrb2-HaloTag, pCAGGS-mEGFP-Sos1, pCAGGS-FLAG-p85-HaloTag, pCAGGS-mEGFP-p110␣, pCXN2-HaloTag-HRasdCT-G12V, pCAGGS- FLAG-BRaf-mEGFP, pCAGGS-FLAG-BRaf-S364A-mEGFP, pCAGGS-FLAG-CRaf-mEGFP, pCAGGS-FLAG-CRaf-S259A-mEGFP, pCXN2-HaloTag-RSK1, pCXN2-HaloTag-RSK2, and pCXN2-HaloTag-RSK3, respectively. Human MEK1, human MEK2, human ERK1, and human ERK2 were obtained by reverse transcription-PCR (RT-PCR) with a HeLa cDNA library and subcloned into the mEGFP or HaloTag vector to generate pCAGGS-FLAG-MEK1-HaloTag, pCAGGS-FLAG-MEK2-HaloTag, pCXN2-mEGFP-ERK1, and pCXN2-mEGFP-ERK2, respectively.…”

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

“…The exact nature of negative feedback loops is not understood, and there appear to be differences between lymphoid and nonlymphoid (epithelial) cell lineages (42)(43)(44)(45). To explore the negative feedback to SOS, we modified our model to include these parameters (46), the phosphorylation rate of SOS1 by pERK, k p ϭ 0.02 s Ϫ1 M Ϫ1 , and the dephosphorylation rate of pSOS1, k d . The process of dephosphorylation of pSOS1 is extremely slow; we therefore take k d as 0 s these feedback loops does not change the overall qualitative behavior of our stochastic models that explore the wild-type, no-SOS1, and lack-of-allosteric-activation-of-SOS situations.…”

Biology using engineering tools

“…First, according to theory, when negative feedback is present, the relationship between system output and negative feedback. 39 This would afford a simple mechanism for a dosage dependent negative feedback. Gab1, a scaffolding protein involved in PI-3K and RasGEF recruitment to the plasma membrane, is also inhibited by ERK-dependent phosphorylation.…”