Quantitative Studies of Signaling Pathways

LAUREN MCNEILL; PENG JIANG; ALEJANDRA C VENTURA; SOFIA D MERAJVER; ALEXANDER J NINFA

Ann Arbor, USA

Simposio; Cellular and Molecular Biology 28th Annual Symposium; 2008

Department of Cellular and Molecular Biology

Signaling pathways have been studied qualitatively for some time, but in order to understand the dynamics of the systems, a more quantitative approach is necessary.  Our labs studied the well-characterized nitrogen assimilation cycle in Escherichia Coli, using an in vitro reconstituted system.  We used α-32P labeled UTP to measure quantitatively the covalent modifications of the signal transduction protein PII in E. Coli over time. PII is a homotrimer modified by the addition of up to three uridylyl groups.  The addition and removal of uridylyl groups on PII is catalyzed by a single enzyme with both uridylyl transferase (UT) and uridylyl removing (UR) activities.The modification of PII is regulated by small molecule effectors glutamine and α-ketoglutarate.  Glutamine regulates the modification of PII by activating the UR activity and inhibiting the UT activity of the UR/UT enzyme.  Small molecule α-ketoglutarate acts by directly binding the PII trimer.  Up to three α-ketoglutarate molecules can bind to the PII protein, but after the first molecule is bound, further binding displays negative cooperativity.  Under conditions of low α-ketoglutarate, when about one molecule per trimer is bound, PII is more likely to be in an uridylylated state (PII-UMP).  Under saturating conditions of α-ketoglutarate, PII is more likely to be in an unmodified state (PII).  Thus it has been theorized that when one molecule of α-ketoglutarate binds, it brings about a conformational change such that PII is activated toward modification.  When PII is saturated with α-ketoglutarate however, another conformational change is brought about that makes modification difficult.In addition to the quantitative experiments that were performed, extensive modeling utilizing ordinary differential equations was completed, in an effort to understand the effects of signaling on downstream targets.  In addition, retroactivity, or the backward flow of information from downstream targets to proteins upstream of the pathway was studied.  Retroactivity has been previously theorized to exist, but these experiments were the first of their kind to quantify the effect of retroactivity on an in vitro system.  In order to study retroactivity in this pathway, the PII/PII-UMP and UT/UR monocycle was allowed to reach steady state, and then, downstream protein NRII was added.  NRII is a dimer protein that can be autophosphorylated when ATP is present.  NRII can bind to PII, thus sequestering it away from the PII/PII-UMP and UT/UR monocycle.  By studying this phenomenon, we were able to quantify retroactive behavior.