PDK1 regulates B cell differentiation and homeostasis.

Successful B cell differentiation and prevention of cell transformation depends on balanced and fine-tuned activation of cellular signaling pathways. The phosphatidyl inositol-3 kinase (PI3K) signaling pathway has emerged as a major regulator of B lymphocyte homeostasis and function. Phosphoinositide-dependent protein kinase-1 (PDK1) is the pivotal node in the PI3K pathway, regulating the stability and activity of downstream AGC kinases (including Akt, RSK, S6K, SGK, and PKC). Although the importance of PI3K activity in B cell differentiation is well documented, the role of PDK1 and other downstream effectors is underexplored. Here we used inducible and stage-specific gene targeting approaches to elucidate the role of PDK1 in early and peripheral B cell differentiation. PDK1 ablation enhanced cell cycle entry and apoptosis of IL-7-dependent pro-B cells, blocking Ig synthesis and B cell maturation. PDK1 also was essential for the survival and activation of peripheral B cells via regulation of PKC and Akt-dependent downstream effectors, such as GSK3α/ß and Foxo1. We found that PDK1 deletion strongly impaired B cell receptor (BCR) signaling, but IL-4 costimulation was sufficient to restore BCR-induced proliferation. IL-4 also normalized PKCß activation and hexokinase II expression in BCR-stimulated cells, suggesting that this signaling pathway can act independent of PDK1 to support B cell growth. In summary, our results demonstrate that PDK1 is indispensable for B cell survival, proliferation, and growth regulation.

Characterization of fragmented 3-phosphoinsitide-dependent protein kinase-1 (PDK1) by phosphosite-specific antibodies.

AIMS: The 3-phosphoinositide-dependent protein kinase-1 (PDK1) activates a number of protein kinases of the AGC subfamily, including protein kinase B and ribosomal S6 protein kinase by phosphorylating these kinases at the activation-loop. PDK1 activity is regulated by auto-phosphorylation and is further increased by stimulation of cells. PDK1 has been shown to have several phosphorylation sites including 5 serine and 3 tyrosine residues. However, Ser241 and Tyr373/376 are only involved in the regulation of PDK1 activity. MAIN METHODS: In this study, we found the putative fragments of PDK1 by using anti-Myc and anti-PDK1 antibodies. Furthermore, the existence of four different sizes of PDK1 were confirmed with other phosphosite specific antibodies. KEY FINDINGS: Taken together, the catalytic domain of PDK1 (42 kDa and 37 kDa) is separately existed in the cells and might be important for the regulation of subset of PDK1 substrate. Because the crystal structural studies suggested that PIF-pocket is located at the catalytic domain and plays a critical role on substrate recognition. SIGNIFICANCE: These suggested importance and roles of this fragment are needed to be determined. Further study on these fragments of PDK1 will provide new insight on the regulatory mechanism of PDK1 in patho-physiological condition.