Abstract
BackgroundPim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases. Over-expression of Pim-1 and Pim-2 in mice promotes the development of lymphomas, and up-regulation of Pim expression has been observed in several human cancers.ResultsHere we show that the pim kinases are constitutively active when expressed in HEK-293 cells and are able to phosphorylate the Bcl-2 family member Bad on three residues, Ser112, Ser136 and Ser155 in vitro and in cells. In vitro mapping showed that Pim-2 predominantly phosphorylated Ser112, while Pim-1 phosphorylated Ser112, but also Ser136 and Ser155 at a reduced rate compared to Ser112. Pim-3 was found to be the least specific for Ser112, and the most effective at phosphorylating Ser136 and Ser155. Pim-3 was also able to phosphorylate other sites in Bad in vitro, including Ser170, another potential in vivo site. Mutation of Ser136 to alanine prevented the phosphorylation of Ser112 and Ser155 by Pim kinases in HEK-293 cells, suggesting that this site must be phosphorylated first in order to make the other sites accessible. Pim phosphorylation of Bad was also found to promote the 14-3-3 binding of Bad and block its association with Bcl-XL.ConclusionAll three Pim kinase family members predominantly phosphorylate Bad on Ser112 and in addition are capable of phosphorylating Bad on multiple sites associated with the inhibition of the pro-apoptotic function of Bad in HEK-293 cells. This would be consistent with the proposed function of Pim kinases in promoting cell proliferation and preventing cell death.
Highlights
Pim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases
Pim-1 was first identified as an oncogenic gene in murine T-cell lymphomas, which became activated after pro-viral insertion in the 3'UTR of the Pim-1 gene [3]
Pim kinases phosphorylate Bad on three sites in vitro
Summary
Pim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases. Pim-1, 2 and 3 make up a group of closely related mammalian serine/threonine kinases that form part of the calmodulin dependent protein kinase family [1,2]. Pim-1 was first identified as an oncogenic gene in murine T-cell lymphomas, which became activated after pro-viral insertion in the 3'UTR of the Pim-1 gene [3]. Insertion in this region stabilised the mRNA for Pim-1 up-regulating the protein [4]. A second related gene, Pim-2, was identified as a common site of viral insertion in murine lymphomas [7].
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