cAMP activates PKCζ in hematopoietic progenitor cells. (A) PKCζ phosphorylation and expression levels in G2 cells cultured for 24 hours either untreated (CTRL) or in the presence of 500 μM dbcAMP (cAMP), as assayed by immunoblot. PKCζ-phosphorylated (P-PKCζ) level was determined by densitometry, normalized to the PKCζ protein level (Total), and expressed as fold change versus CTRL cells. Results are mean ± SD of 5 independent experiments. (B) PKCζ membranal translocation in cAMP-treated cells. MPBL CD34⁺ cells were transfected with GFP (green fluorescent protein) only (top and middle rows) or with Spa1GFP (bottom row) expressing vector and further incubated for 18 hours either untreated (CTRL) or in the presence of 500 μM cAMP and indirectly immunolabeled for PKCζ (red). GFP vector transfected cells were colabeled for CXCR4 (green) because no GFP signal was retained after the permeabilization procedure. Note the increased CXCR4 expression and colocalization with PKCζ at the membrane of cAMP-stimulated cells (middle row). Spa1GFP overexpression decreased the proportion of cells displaying membranal PKCζ labeling (histogram on the right, representative of 2 independent experiments; 250 cells in each sample), yet PKCζ translocation to the membrane could be detected in Spa1GFP-transfected cells (bottom row, asterisks). Bar represents 10 μm. (C) Inhibition of the cAMP-induced PKCζ phosphorylation (P-PKCζ) by the NSC23766 (Rac1 inh) in G2 cells treated and assayed as in panel A. Results of the densitometry analysis, expressed in arbitrary units (AU), are represented as mean ± SD of 3 independent experiments.