Figure 3

Transient activation of PKA by incoming Ad2 requires cell surface integrins. Drug-treated or control HeLa cells were incubated in the cold with 6 10⁴ Ad2 particles per cell (or as indicated) for 1 h, washed with virus-free medium and warmed up for different times. Kemptide phosphorylations (representing PKA activity) were determined in cell lysates at 15 min p.i. and normalized to the total cAMP-induced kemptide phosphorylations. (A) PKA stimulation by Ad2 is transient and peaks at 15 min p.i. Phospho-kemptide amounts are indicated as the mean values (left axis) including the SEM and number of independent experiments (n). Fold activation of infected (dark bars) over non-infected (light bars) cells is indicated by the solid line. (B) Ad2-stimulated PKA activation amounts to 3% of the total cellular PKA activity and is comparable to the levels in forskolin-stimulated HeLa cells, which can be boosted by the diesterase inhibitor IBMX. Recombinant PKI added to the cell lysates reduced both Ad2- and cAMP-stimulated kemptide phosphorylations to near background levels. (C) PKA activation is dependent on Ad2 particle dose (p/cell) and diminished by pre-incubating cells with the PKA inhibitor H89. The Ad2 mutant ts1 fails to stimulate kemptide phosphorylation. (D) cRGD or low-calcium medium, but not the PKC inhibitor calphostin (calph), the MEK inhibitor PD or nocodazole block Ad2-induced kemptide phosphorylation.