Fig. 2.

Type I PKA is resolved in cytosolic microtubule fractions of PDE4D4_1–166-expressing PMVECs. A: immunoblotting revealed an increase in the PKA catalytic subunit in the cytosolic fractions of PDE4D4_1–166-expressing cells compared with vector control cells. This increase in the PKA catalytic subunit observed in the cytosolic fraction was paralleled by a decrease in the membrane fraction of PDE4D4_1–166-expressing cells. Similarly, the type I regulatory subunit of PKA was enriched in the cytosolic fractions and absent in the membrane fractions of PDE4D4_1–166-expressing cells compared with controls. Forskolin (1 μM for 30 min) treatment did not further change the amounts of catalytic or regulatory proteins in either the cytosol or membrane fractions. PDE4D4_1–166 expression did not change the β-tubulin levels in the cytosol of PMVECs. Immunofluorescence shows the PKA catalytic (B, red) and type I regulatory (C, red) subunits colocalize with microtubules (B and C, green) in basal and forskolin-stimulated PDE4D4_1–166-expressing PMVECs. Following forskolin treatment, PKA catalytic and regulatory subunits were enriched in reorganized microtubules. The type II regulatory PKA subunit (red) was primarily localized to the perinuclear region (D) and did not intensely interact with microtubules (green) under basal conditions or after forskolin stimulation in PDE4D4_1–166-expressing PMVECs. For B–D, the white boxes denote the area enlarged on the right of each image set. Colocalization is indicated by yellow in the merged images (arrowheads). E: catalytic and type I regulatory PKA subunits were principally found in the depolymerized pool of microtubules. β-Tubulin was immunoprecipitated (IP), and catalytic and type I regulatory subunits were immunoblotted from either depolymerized or polymerized microtubules. Forskolin activation did not increase the coimmunoprecipitation of PKA subunits with β-tubulin.