Dynamic control of Epac2 localization by cAMP and Ca2+-mediated activation of Ras

• Epac2, a cAMP-regulated guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2, is an important mediator of a variety of cAMP-regulated cellular processes, including insulin secretion from pancreatic β-cells. Epac2 has been suggested to associate with the plasma membrane by interacting with active Ras (Ras-GTP), but the dynamics and regulation of membrane binding is unknown. Using real-time confocal and total internal reflection fluorescence microscopy we demonstrate that cAMP-elevating agents cause rapid translocation of GFP-tagged Epac2 from the cytoplasm to the plasma membrane in insulin-secreting MIN6 β-cells. Glucose concentrations that stimulate insulin secretion often triggered oscillatory translocation of GFP-Epac2 following oscillations of the sub-membrane concentrations of cAMP and Ca2+ ([cAMP]pm and [Ca2+]pm). The translocation was suppressed after inhibition of adenylyl cyclases or removal of extracellular Ca2+. GFP-Epac2 translocation by rise of [Ca2+]pm required concomitant elevation of [cAMP]pm and cAMP-induced translocation was enhanced by moderate [Ca2+]pm elevations. However the effect of Ca2+ was dual since translocation was inhibited by high [Ca2+]pm spikes. Epac2 mutants lacking the cAMP-binding or Ras-association domains were unable to translocate and localized constitutively to the plasma membrane and cytoplasm, respectively. Ras activity monitored with a fluorescent Ras-GTP binding reporter was tightly correlated with the translocation of Epac2. It is concluded that Epac2 localization is dynamically controlled by cAMP as well as by Ca2+-mediated activation of Ras, and that reversible translocation of Epac2 between the cytoplasm and plasma membrane requires both Ras-association and cAMP-binding domains. Spatio-temporal control of Epac2 in β-cells has implications for the understanding of its involvement in insulin secretion kinetics by Rap GTPases and other downstream effectors at the plasma membrane.

Subject headings

NATURVETENSKAP  -- Biologi -- Cellbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Cell Biology (hsv//eng)

Keyword

cAMP

Ca2

Epac2

Ras

beta-cell

evanescent wave microscopy

glucose

Cell biology

Cellbiologi

Medical Cell Biology

Medicinsk cellbiologi

Publication and Content Type

vet (subject category)

ovr (subject category)