Abstract
We have reported previously the cloning and characterization of human and mouse protein kinase B gamma (PKB gamma), the third member of the PKB family of second messenger-regulated serine/threonine kinases (Brodbeck, D., Cron, P., and Hemmings, B. A. (1999) J. Biol. Chem. 274, 9133--9136). Here we report the isolation of human and mouse PKB gamma 1, a splice variant lacking the second regulatory phosphorylation site Ser-472 in the hydrophobic C-terminal domain. Expression of PKB gamma 1 is low compared with PKB gamma, and it is regulated in different human tissues. We show that PKB gamma and PKB gamma 1 differ in their response to stimulation by insulin, pervanadate, peroxide, or okadaic acid. Activation of PKB gamma 1 requires phosphorylation at a single regulatory site Thr-305. Interestingly, this site is phosphorylated to a higher extent in PKB gamma compared with PKB gamma 1 upon maximal stimulation by pervanadate, and this is reflected in the respective specific kinase activities. Furthermore, upon insulin stimulation of transfected cells, PKB gamma 1 translocates to the plasma membrane to a lesser extent than PKB gamma. Taken together, these results suggest that phosphorylation of the hydrophobic motif at the extreme C terminus of PKB gamma may facilitate translocation of the kinase to the membrane and/or its phosphorylation on the activation loop site by phosphoinositide-dependent protein kinase-1.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY005799
These results suggest that phosphorylation of the hydrophobic motif at the extreme C terminus of Protein kinase B (PKB)␥ may facilitate translocation of the kinase to the membrane and/or its phosphorylation on the activation loop site by phosphoinositide-dependent protein kinase-1
Previous studies on the regulation of the three PKB isoforms ␣, , and ␥ revealed the central importance of the two regulatory phosphorylation sites, Thr-308/309/305 and Ser-473/474/ 472, in controlling kinase activity in response to insulin or growth factor signaling via the phosphoinositide 3-kinase pathway, with both sites required for full activation
Summary
Cloning and Construction of Expression Vectors HA-PKB␥, HAPKB␥1, and Phosphorylation Site Mutants—The cloning of human and mouse HA-PKB␥ has been described previously [7]. Reverse Transcription and Semiquantitative PCR—Reverse transcription was performed with the GeneAmp RNA PCR kit (PE Biosystems), essentially according to the manufacturer’s instructions, using random primers and 1 g of total RNA from mouse brain or embryos as template in a total volume of 20 l, and a reaction protocol of 10 min at 25 °C, 60 min at 42 °C and 5 min at 99 °C. The cells were washed once with phosphate-buffered saline, fixed in 4% p-formaldehyde (Merck) in phosphate-buffered saline for 30 min at room temperature, and permeabilized with 0.2% Triton X-100 (Sigma) for 5 min They were stained with the monoclonal anti-HA antibody 12CA5 and a fluorescein isothiocyanate-coupled anti-mouse IgG secondary antibody (1:500) and observed by confocal microscopy.
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