Kinetic Analysis of Platelet-derived Growth Factor Receptor/Phosphoinositide 3-Kinase/Akt Signaling in Fibroblasts

Abstract

Isoforms of the serine-threonine kinase Akt coordinate multiple cell survival pathways in response to stimuli such as platelet-derived growth factor (PDGF). Activation of Akt is a multistep process, which relies on the production of 3 -phosphorylated phosphoinositide (PI) lipids by PI 3-kinases. To quantitatively assess the kinetics of PDGF receptor/PI 3-kinase/Akt signaling in fibroblasts, a systematic study of this pathway was performed, and a mechanistic mathematical model that describes its operation was formulated. We find that PDGF receptor phosphorylation exhibits positive cooperativity with respect to PDGF concentration, and its kinetics are quantitatively consistent with a mechanism in which receptor dimerization is initially mediated by the association of two 1:1 PDGF/PDGF receptor complexes. Receptor phosphorylation is transient at high concentrations of PDGF, consistent with the loss of activated receptors upon endocytosis. By comparison, Akt activation responds to lower PDGF concentrations and exhibits more sustained kinetics. Further analysis and modeling suggest that the pathway is saturated at the level of PI 3-kinase activation, and that the p110␣ catalytic subunit of PI 3-kinase contributes most to PDGFstimulated 3 -PI production. Thus, at high concentrations of PDGF the kinetics of 3 -PI production are limited by the turnover rate of these lipids, while the Akt response is additionally influenced by the rate of Akt deactivation.Platelet-derived growth factor (PDGF) 1 is a polypeptide mitogen of broad specificity, one of the earliest and most potent serum factors to be isolated (1). Beyond signaling of proliferation, PDGF acts as a strong chemoattractant during wound healing and can mediate protection from apoptosis in response to serum withdrawal and certain stress stimuli (2, 3). Three forms of PDGF have been studied extensively. They are composed of disulfide-bonded homo-and heterodimers of A and B chains, of which PDGF-BB is the best characterized. There are two structurally related PDGF receptors, ␣ and ␤, which exhibit different affinities for the A chain but roughly equivalent affinities for the B chain (4, 5). More recently, PDGF-C and -D isoforms, which form homodimers, have also been identified; these too exhibit different affinities for PDGF ␣-and ␤-receptors (6 -8).PDGF receptors belong to the well studied class of signal transducers collectively known as receptor-tyrosine kinases (9, 10). As with other receptors of this class, ligand-induced dimerization of PDGF receptors activates their intrinsic kinase domains, which catalyze the autophosphorylation of the receptors on multiple intracellular tyrosine residues in trans (11-13). The phosphorylated receptor may then act as a scaffold for specific binding interactions with cytosolic signal transduction enzymes and adaptor proteins (14). Among the most important of these are isoforms of phosphoinositide (PI) 3-kinase, which phosphorylate phosphatidylinositol (PtdIns) lipids in cell membranes to produce the 3Ј-PI second messengers PtdI...

Mapping growth-factor-modulated Akt signaling dynamics

“…Variable conclusions about population responses to different growth factors have been reached by more traditional endpoint assays, including the use of serial immunoblotting or immunocytochemistry to probe for Akt or substrate phosphorylation (Borisov et al, 2009;Chen et al, 2009;Kubota et al, 2012;Park et al, 2003). Here, by continuous monitoring, we were able to collect a consistent and more robust data set with less experimental effort and fewer assumptions.…”

Adenoviral vector saturates Akt pro-survival signaling and blocks insulin-mediated rescue of tumor-necrosis-factor-induced apoptosis

“…We showed that this is partly due to saturation of Akt-effector phosphorylation, but it remains unclear why saturation occurs. Saturation of the PI3K-Akt pathway has also been reported in other contexts -for instance, in response to high concentrations of certain growth factors (Park et al, 2003) -suggesting that saturation is not Adv-specific. The simplest molecular explanation for saturation in Adv-infected cells is that Akt and its substrates are negatively regulated by different protein phosphatases.…”

Activity of PLCε contributes to chemotaxis of fibroblasts towards PDGF

“…The key network nodes and links include PI3K mediated activation of PKB, activation of Ras and downstream kinases such as ERK and activation of PLCc1 (Park et al, 2003). Several other small GTPases in addition to Ras, such as Rap, Rho and Rac, also participate in responses to PDGF-BB.…”