Abstract
The chemoattractant cAMP, acting through serpentine cAMP receptors, results in a rapid and transient stimulation of the Dictyostelium mitogen-activated protein kinase ERK2 activity (). In this study we show that other pathways required for aggregation, including Ras and cAMP-dependent protein kinase (PKA), are important regulators of ERK2 activation and adaptation. By examining both the level and kinetics of activation and adaptation of ERK2, we show that Ras is a negative regulator of ERK2. Activated Ras or disruption of a Ras GAP gene results in reduced ERK2 activation whereas disruption of putative Ras GEF or expression of dominant negative Ras proteins have a more rapid, higher, and extended activation. CRAC, a PH domain-containing protein required for adenylyl cyclase activation, is also required for proper ERK2 adaptation. PKA overexpression results in a more rapid, higher level of activation, whereas pka null cells show a lower level but more extended ERK2 activation. Furthermore, we show that constitutive expression of PKA catalytic subunit bypasses the requirement of ERK2 for aggregation and later development, indicating that PKA lies downstream from ERK2 and that ERK2 may regulate one or more components of the signaling pathway required for mediating PKA function, possibly by directly regulating PKA R or a protein controlling the intracellular level of cAMP.
Highlights
The chemoattractant cAMP, acting through serpentine cAMP receptors, results in a rapid and transient stimulation of the Dictyostelium mitogen-activated protein kinase ERK2 activity [1]
Ras Is a Negative Regulator of ERK2—The putative Ras GEF Aimless (AleA) is required for proper aggregation of Dictyostelium cells [10]. aleA null cells show normal activation of guanylyl cyclase but have a significantly reduced level of adenylyl cyclase activation in response to cAMP [10], as has been shown for erk2 null cells [8]
To determine whether Aimless, and by implication a Ras protein, was involved in the regulation of ERK2 activation, we examined the kinetics and level of activation of ERK2 in an aimless null strain using an in gel assay in which an ERK2 substrate, myelin basic protein (MBP), is added into the SDS gel
Summary
The chemoattractant cAMP, acting through serpentine cAMP receptors, results in a rapid and transient stimulation of the Dictyostelium mitogen-activated protein kinase ERK2 activity [1]. During the preaggregation and aggregation stages of Dictyostelium development, the MAP kinase ERK2 is activated through cAMP and folate chemotactic receptors [1].2 Stimulation with extracellular cAMP results in a Ͼ40-fold increase in ERK2 activity that peaks at ϳ50 s and thereupon adapts, reaching its basal level within 5– 8 min.
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