Common Polymorphism in the Phosphatase PHLPP2 Results in Reduced Regulation of Akt and Protein Kinase C

Gloria Reyes,Noel Warfel,Matthew Niederst,Alexandra C Newton,John Brognard

doi:10.1074/jbc.m901468200

Gloria Reyes, Noel Warfel + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m901468200

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Abstract

PHLPP2 (PH domain leucine-rich repeat protein phosphatase 2) terminates Akt and protein kinase C (PKC) activity by specifically dephosphorylating these kinases at a key regulatory site, the hydrophobic motif (Ser-473 in Akt1). Here we identify a polymorphism that results in an amino acid change from a Leu to Ser at codon 1016 in the phosphatase domain of PHLPP2, which reduces phosphatase activity toward Akt both in vitro and in cells, in turn resulting in reduced apoptosis. Depletion of endogenous PHLPP2 variants in breast cancer cells revealed the Ser-1016 variant is less functional toward both Akt and PKC. In pair-matched high grade breast cancer samples we observed retention of only the Ser allele from heterozygous patients (identical results were observed in a pair-matched normal and tumor cell line). Thus, we have identified a functional polymorphism that impairs the activity of PHLPP2 and correlates with elevated Akt phosphorylation and increased PKC levels.

Highlights

Identification of a Polymorphism in PHLPP2—To determine if the PHLPP2 phosphatase is mutated in breast cancer, we revealed that the T-ϾC nucleotide change at position 3047 is a polymorphism present in 30% of the population
The absence of the Ser/Ser genotype in the control population, but its presence in breast cancer cell lines, led us to ask whether this polymorphism could play a role in breast tumorigenesis
We did not observe loss of either allele in the five grade 2 breast cancer samples (Fig. 5, A and C). These data suggest that preferential loss of the Leu allele in high grade breast cancers may contribute to the aggressive phenotype of these cancers by decreasing the basal phosphatase activity of PHLPP2, resulting in an increase in Akt phosphorylation, protein kinase C (PKC) protein levels, or both

Summary

EXPERIMENTAL PROCEDURES

Materials—SMARTpool siRNAs against PHLPP1 and PHLPP2 were purchased from Dharmacon. The following antibodies were purchased from Cell Signaling: phospho-specific to Thr-308 and Ser-473 of Akt, Akt, PTEN, and ERK1/2. SiRNA transfections were performed using 50 nM SMARTpool siRNA, and cells were incubated for 48 h and either lysed or their media was changed to low serum conditions for 12 h and EGF (10 ng/ml) was added for the indicated times prior to lysis. Transfected cells were lysed in Buffer 1 (50 mM Na2HPO4, pH 7.5, 1 mM sodium pyrophosphate, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, 1% SDS, 1 mM DTT, 200 ␮M benzamidine, 40 ␮g mlϪ1 leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and sonicated for 5 s. The pellet (containing the nuclear fraction) was resuspended in 200 ␮l of membrane buffer (50 mM Na2HPO4, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 1 mM sodium pyrophosphate, 20 mM NaF, 1 mM DTT, 200 ␮M benzamidine, 40 ␮g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride), and centrifuged at 16,000 ϫ g for 15 min at 4 °C. The pellet was resuspended in 200 ␮l of membrane buffer and centrifuged at 108,920 ϫ g for 20 min at 4 °C; the resulting supernatant was the membrane fraction

RESULTS

Ser allele in the remaining breast

DISCUSSION