FIGURE 4.

Spatial and temporal regulation of the localization of PdkA and PdkB. A, the subcellular localization, translocation activities, and chemoattractant-induced modification of a series of deletions of PdkA are summarized. PM, plasma membrane; +, −, or ND means positive, no response, or not determined for chemoattractant-induced translocation or modification, respectively. B, localization of PdkA-(1–686)-GFP and PdkA-(1–483)-GFP were shown in wild-type (WT) cells during chemotaxis. C, fluorescent images of cells expressing GFP and a series of PdkA deletion proteins fused with GFP are shown. D, localizations of PdkA-(1–686)-GFP or PdkA-(477–686)-GFP are compared in wild-type and pi3k¹⁻²⁻ cells. E, to evaluate translocation activity, wild-type cells expressing PdkA-GFP or PdkB-GFP were stimulated with 1 μm cAMP, and fluorescent images were taken every 2 s. Representative responses are shown by a series of time-lapsed images. F, wild-type and pi3k¹⁻²⁻ cells were stimulated with 1 μm cAMP and lysed at the indicated times. The membrane-enriched fraction was collected by centrifugation and probed with α-GFP antibody (upper panel) and α-phospho-protein kinase C (pan) antibody (middle panel). Coomassie Brilliant Blue (CBB) staining of the membrane for the immunoblotting was used as a loading control. Scale bars represent 10 μm.