Abstract
By studying primary isogenic murine embryonic fibroblasts (MEFs), we have shown that PLK3 null MEFs contain a reduced level of phosphatase and tensin homolog (PTEN) and increased Akt1 activation coupled with decreased GSK3β activation under normoxia and hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, efficiently phosphorylates PTEN in vitro. Mass spectrometry identifies threonine 366 and serine 370 as two putative residues that are phosphorylated by Plk3. Immunoblotting using a phosphospecific antibody confirms these sites as Plk3 phosphorylation sites. Moreover, treatment of MEFs with LiCl, an inhibitor of GSK3β and CK2, only partially suppresses the phosphorylation, suggesting Plk3 as an additional kinase that phosphorylates these sites in vivo. Plk3-targeting mutants of PTEN are expressed at a reduced level in comparison with the wild-type counterpart, which is associated with an enhanced activity of PDK1, an upstream activator of Akt1. Furthermore, the reduced level of PTEN in PLK3 null MEFs is stabilized by treatment with MG132, a proteosome inhibitor. Combined, our study identifies Plk3 as a new player in the regulation of the PI3K/PDK1/Akt signaling axis by phosphorylation and stabilization of PTEN.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Adjuvants, Immunologic / pharmacology
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Animals
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Cysteine Proteinase Inhibitors / pharmacology
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Embryo, Mammalian / cytology
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Embryo, Mammalian / metabolism
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Enzyme Stability / drug effects
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Enzyme Stability / genetics
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Fibroblasts / cytology
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Fibroblasts / metabolism
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Glycogen Synthase Kinase 3 / genetics
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Glycogen Synthase Kinase 3 / metabolism
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Glycogen Synthase Kinase 3 beta
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HEK293 Cells
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Humans
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Leupeptins / pharmacology
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Lithium Chloride / pharmacology
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Mice
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Mice, Knockout
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Mutation
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PTEN Phosphohydrolase / genetics
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PTEN Phosphohydrolase / metabolism*
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Phosphorylation / drug effects
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Phosphorylation / genetics
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Proto-Oncogene Proteins c-akt / genetics
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Proto-Oncogene Proteins c-akt / metabolism
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Pyruvate Dehydrogenase Acetyl-Transferring Kinase
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Tumor Suppressor Proteins
Substances
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Adjuvants, Immunologic
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Cysteine Proteinase Inhibitors
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Leupeptins
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PDK1 protein, human
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Pdk1 protein, mouse
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Pyruvate Dehydrogenase Acetyl-Transferring Kinase
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Tumor Suppressor Proteins
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PLK3 protein, human
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AKT1 protein, human
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Akt1 protein, mouse
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GSK3B protein, human
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Glycogen Synthase Kinase 3 beta
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Gsk3b protein, mouse
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Plk3 protein, mouse
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Glycogen Synthase Kinase 3
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PTEN Phosphohydrolase
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PTEN protein, human
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Pten protein, mouse
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Lithium Chloride
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benzyloxycarbonylleucyl-leucyl-leucine aldehyde