Determinants of the tumor suppressor INPP4B protein and lipid phosphatase activities

Highlights

• INPP4B has protein tyrosine phosphatase activity.

• INPP4B active site residues play important roles in regulating lipid, protein or both phosphastase activities.

• Conserved residues between the INPP4B and PTEN active sites confer distinct properties to each protein.

• INPP4B can suppress activation of Akt through dephosphorylation of Akt tyrosine residues.

Abstract

The tumor suppressor INPP4B is an important regulator of phosphatidyl-inositol signaling in the cell. Reduced INPP4B expression is associated with poor outcomes for breast, prostate, and ovarian cancer patients. INPP4B contains a CX₅R catalytic motif characteristic of dual-specificity phosphatases, such as PTEN. Lipid phosphatase activity of INPP4B has previously been described. In this report we show that INPP4B can dephosphorylate para-nitrophenyl phosphate (pNPP) and 6,8-difluoro-4-methylumbelliferyl (DiFMUP), synthetic phosphotyrosine analogs, suggesting that INPP4B has protein tyrosine phosphatase (PTP) activity. Using mutagenesis, we examined the functional role of specific amino acids within the INPP4B C₈₄₂KSAKDR catalytic site. The K843M mutant displayed increased pNPP hydrolysis, the K846M mutant lost lipid phosphatase activity with no effect on PTP activity, and the D847E substitution ablated PTP activity and significantly reduced lipid phosphatase activity. Further, we show that INPP4B but not PTEN is able to reduce tyrosine phosphorylation of Akt1 and both the lipid and PTP activity of INPP4B likely contribute to the reduction of Akt1 phosphorylation. Taken together our data identified key residues in the INPP4B catalytic domain associated with lipid and protein phosphatase activities and found a robust downstream target regulated by INPP4B but not PTEN.

Introduction

Deregulation of phosphatidyl inositol signaling plays an important role in various disorders. These pathways are stimulated by phosphatidyl 3, 4, and 5 kinases (PI3K, PI4K, PI5K) and inhibited by lipid phosphatases such as PTEN, INPP4B, and SHIP [1]. These phosphatases dephosphorylate the phosphatidyl inositol ring on the 3rd, 4th, and 5th position respectively. Loss of INPP4B is a poor prognostic factor for breast, ovarian, and prostate cancers [2], [3], [4]. Similar to PTEN, INPP4B contains a dual specificity phosphatase (DuSP) domain with a characteristic DuSP CX₅R motif, C₈₄₂KSAKDR₈₄₈. Residue C₈₄₂ is required for INPP4B enzymatic activity [3], [5]. There are three known INPP4B substrates: phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) [5]. Substrates of INPP4B lipid phosphatase activity are important second messengers in pathways regulating cellular proliferation and metastasis and have been implicated in prostate cancer progression [6], [7]. PI(4,5)P2 is a substrate for both INPP4B and phospholipase C, an enzyme implicated in cellular motility and tumor dissemination in cancer [8], [9]. PI(3,4)P2 is present at low levels on the cell membrane and accumulates at the sites of invadopodia [10], cellular appendages responsible for focal pericellular proteolysis of the extracellular matrix necessary for cellular invasion and metastasis [11]. PI(3,4)P2 binds to the pleckstrin homology domains of Akt and PDK1, recruiting them to the plasma membrane, leading to Akt phosphorylation on T308 and S473, and the activation of Akt. In addition to T308 and S473, tyrosine (Y) phosphorylation of Akt is emerging as an important regulatory mechanism for Akt signaling. Recruitment to the cell membrane and activation of Akt are regulated by its phosphorylation on several Y residues [12], [13], [14]. Elevated Y176 phosphorylation promotes Akt recruitment to the plasma membrane and increases phosphorylation of the Akt residues T308 and S473 [12]. In breast cancer patients, increased levels of Y176 phosphorylation correlate with poor overall survival [13].

In this report, we show that INPP4B can dephosphorylate para-nitrophenyl phosphate (pNPP) and 6,8-difluoro-4-methylumbelliferyl (DifMUP), analogs of phosphotyrosine, suggesting that INPP4B has protein tyrosine phosphatase (PTP) activity. Mutation of four key residues in the active site identified distinct residues that contribute to INPP4B lipid and protein phosphatase activities. Finally, we found that INPP4B reduced Akt1 phospho-Y levels in Hek293T cells. Co-expression of Akt1 with wild-type (WT) and mutant INPP4B proteins revealed that both lipid and PTP activities contribute to downregulation of Akt1 phospho-Y levels. PTEN overexpression did not reduce phosphorylation of Akt1 on Y residues, suggesting distinct downstream signaling for INPP4B and PTEN tumor suppressors.