Regulation of insulin receptor signaling by the protein tyrosine phosphatase TCPTP

Abstract

The human protein tyrosine phosphatase TCPTP exists as two forms: an endoplasmic reticulum-targeted 48-kDa form (TC48) and a nuclear 45-kDa form (TC45). Although targeted to the nucleus, TC45 can exit in response to specific stimuli to dephosphorylate cytoplasmic substrates. In this study, we investigated the downregulation of insulin receptor (IR) signaling by TCPTP. In response to insulin stimulation, the TC48-D182A and TC45-D182A "substrate-trapping" mutants formed stable complexes with the endogenous tyrosine-phosphorylated IR beta-subunit in 293 cells. Moreover, in response to insulin stimulation, the TC45-D182A mutant accumulated in the cytoplasm of cells overexpressing the IR and in part colocalized with the IR beta-subunit at the cell periphery. These results indicate that the IR may serve as a cellular substrate for both TC48 and TC45. In immortalized TCPTP(-/-) murine embryo fibroblasts, insulin-induced IR beta-subunit tyrosine phosphorylation and protein kinase PKB/Akt activation were enhanced relative to the values in TCPTP(+/+) cells. Importantly, the expression of TC45 or TC48 to physiological levels suppressed the enhanced insulin-induced signaling in TCPTP(-/-) cells. These results indicate that the differentially localized variants of TCPTP may dephosphorylate the IR and downregulate insulin-induced signaling in vivo.

FIG. 1.

TCPTP recognizes the tyrosine-phosphorylated IR β-subunit as a cellular substrate. 293 cells transfected with vector control or plasmids expressing TC45, TCA5-D182A (TC45D), TC48, or TC48-D182A (TC48D) were serum starved and stimulated with 100 nM insulin (INS) for 15 min. (A) TCPTP immunoprecipitates were resolved by SDS-PAGE and immunoblotted with polyclonal antibodies specific for the IR β-subunit phosphorylated on tyrosines 1162 and 1163 (phospho-IR) or TCPTP. (B) TCPTP immunoprecipitates were resolved and immunoblotted with antibodies specific for pTyr and reprobed for phospho-IR. Immunoblots were then stripped as described previously (46) and reprobed with antibodies specific for the IR β-subunit (IRβ) or TCPTP.

FIG. 2.

TC45-D182A exits the nucleus and colocalizes with IR β-subunit in response to insulin. CHO/IR cells were transfected with pCG constructs expressing wild-type TC45 or the TC45-D182A substrate-trapping mutant (TC45D). At 24 h posttransfection, cells were serum starved and either left untreated or stimulated with 100 nM insulin (INS) for 30 min. Cells were processed for (A) epifluorescence microscopy with the monoclonal anti-TCPTP antibody CF4 or (B) for confocal microscopy with the rabbit polyclonal anti-TCPTP antibody 6228 and a mouse monoclonal IR β-subunit (IRβ) antibody as described previously (48).

FIG. 3.

Expression of signaling proteins mediating insulin action in TCPTP^−/− versus TCPTP^+/+ cells. Equal amounts of protein from TCPTP^−/− (EFM4^−/−) and TCPTP^+/+ (EFM7^+/+) cells were resolved on SDS-PAGE and immunoblotted with anti-TCPTP 3E2 antibody (23) or antibodies specific for PTP1B, the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K), IR β-subunit (IRβ), IRS-1 (indicated by arrow), or actin.

FIG. 4.

Insulin-induced tyrosine phosphorylation in TCPTP^−/− versus TCPTP^+/+ cells. (A) TCPTP^−/− (EFM4^−/−) or TCPTP^+/+ (EFM7^+/+) cells were either left untreated or stimulated with 100 nM insulin (INS) for 10 min. Cells were lysed, and proteins were resolved on SDS-PAGE and immunoblotted with phosphotyrosine (pTyr)-specific antibodies. Molecular mass standards (precision prestained standards; Bio-Rad, Hercules, Calif.) are indicated on the right, and an arrow on the left highlights the ≈95-kDa pTyr protein in TCPTP^−/− cells following insulin stimulation. (B) TCPTP^−/− (EFM4^−/−) or TCPTP^+/+ (EFM7^+/+) cells were serum starved and either left untreated or stimulated with 10 nM insulin for 60 min. Equal amounts of protein were resolved by SDS-PAGE and immunoblotted with antibodies specific for pTyr and then stripped and reprobed with antibodies specific for the IR β-subunit (IRβ). (C) EFM14^−/− or EFM11^+/+ cells were serum starved and either left untreated or stimulated with 10 nM insulin for 60 min. Proteins were resolved and immunoblotted for pTyr and then stripped and reprobed for IR β-subunit. Molecular mass standards (prestained protein markers, broad range; New England Biolabs, Beverly, Mass.) are indicated on the right, and an arrow highlights the ≈95-kDa pTyr protein in TCPTP^−/− cells following insulin stimulation. (D) TCPTP^−/− (EFM4^−/−) or TCPTP^+/+ (EFM7^+/+) cells were serum starved and either left untreated or stimulated with 10 nM insulin for 60 min. Cells were lysed in immunoprecipitation lysis buffer, and the pTyr-containing proteins were immunoprecipitated as described under Materials and Methods. The immunoprecipitates (IPs) and equal quantities of the corresponding lysates prior to immunoprecipitation were resolved by SDS-PAGE and immunoblotted with antibodies specific for IRβ. Representative immunoblots are shown for clone 45-R5 in response to 1 or 10 nM insulin. Quantitations of PKB/Akt phosphorylation in response to insulin stimulation in TCPTP^−/−, 45-R5, 45-R19, and 45-R20 cells are also shown. In each case the phospho-Akt immunoblots were quantitated by densitometric analysis and normalized for total Akt protein in corresponding Akt immunoblots, with the phospho-Akt/Akt ratio in the absence of insulin being set at zero. Units are arbitrary and are means ± standard errors of at least three independent experiments.

FIG. 5.

Enhanced insulin-induced PKB/Akt signaling in TCPTP-null cells. (A) TCPTP^−/− or TCPTP^+/+ cells were serum starved and either left untreated or stimulated with 1 nm (A) or 10 nm (B) insulin for 1 to 60 min. Cells were collected in hot 3× Laemmli sample buffer, and proteins were resolved by SDS-PAGE, transferred onto the same Immobilon-P (Millipore, Bedford, Mass.) membrane, and immunoblotted with antibodies specific for phosphorylated and activated PKB/Akt (Phos-Akt). Immunoblots were then stripped and reprobed with antibodies specific for total PKB/Akt protein. Representative immunoblots are shown. Quantitations of Akt phosphorylation in response to 1 nM or 10 nM insulin stimulation in TCPTP^−/− and TCPTP^+/+ cells are also shown. Phospho-Akt immunoblots were quantitated by densitometric analysis and normalized for total Akt protein in corresponding Akt immunoblots, with the phospho-Akt/Akt ratio in the absence of insulin being set at zero. Units shown are arbitrary and are means ± standard errors of at least three independent experiments.

FIG. 6.

TC48 and TC45 expression in TCPTP^+/+ cells. (A) Equal amounts of protein from TCPTP^−/− (EFM4^−/−) and TCPTP^+/+ (EFM7^+/+) cells were resolved on SDS-PAGE and immunoblotted with polyclonal TCPTP 6228 antibodies. (B) PCR of purified murine TC48 (mTC48) or murine TC45 (mTC45) cDNA (1 pg of either mTC48 or mTC45 cDNA was used in a 25-μl reaction as indicated in Materials and Methods) with oligonucleotide primers specific for either TC45 or TC48 (indicated on the left). (C) Reverse transcription-PCR from TCPTP^+/+ or TCPTP^−/− cells as indicated under Materials and Methods with oligonucleotide primers specific for either TC45 or TC48 (indicated on the left). Water was substituted for first-strand cDNA in negative controls (−), and β-actin reverse transcription-PCRs were used for normalization. PCRs in panels B and C were resolved on agarose gels and visualized by ethidium bromide staining. (D) Comparison of TCPTP expression in TCPTP^+/+ versus TC45-reconstituted (45-R5, 45-R10, 45-R19, and 45-R20) or TC48-expressing (48-R9, 48-R11, and 48-R12) cells. Equal amounts of protein from the indicated cell lines were resolved on SDS-PAGE and immunoblotted with polyclonal TCPTP 6228 antibodies.

FIG. 7.

TC45 reconstitution suppresses the enhanced IR β-subunit tyrosine phosphorylation in TCPTP-null cells. TCPTP^−/−, TCPTP^+/+, or 45-R5 cells were serum starved and either left untreated or stimulated with 10 nM insulin (INS) for 60 min. Cells were lysed, and the phosphotyrosine (pTyr)-containing proteins were immunoprecipitated as described under Materials and Methods. The immunoprecipitates and equal quantities of the corresponding lysates prior to immunoprecipitation were resolved by SDS-PAGE and immunoblotted with antibodies specific for IR β-subunit.

FIG. 8.

TC45 reconstitution suppresses the enhanced insulin-induced PKB/Akt signaling in TCPTP-null cells. TCPTP^−/− and either 45-R5, 45-R19, or 45-R20 cells were serum starved and either left untreated or stimulated with (A) 1 nM or (B) 10 nM insulin for 1 to 60 min. Cells were collected in hot 3× Laemmli sample buffer, and lysates were resolved by SDS-PAGE, transferred onto the same Immobilon-P membrane, and immunoblotted with antibodies specific for phosphorylated and activated PKB/Akt (Phos-Akt) and then stripped and reprobed for PKB/Akt.

FIG. 9.

TC48 expression suppresses enhanced insulin-induced signaling in TCPTP-null cells. (A) TCPTP^−/− or TC48-expressing 48-R9 or 48-R11 cells were serum starved and either left untreated or stimulated with 10 nM insulin for 60 min. Equal amounts of protein were resolved by SDS-PAGE and immunoblotted with antibodies specific for phosphotyrosine (pTyr) and then stripped and reprobed with antibodies specific for the IR β-subunit (IRβ). IR β-subunit in the pTyr immunoblot is indicated by the arrow on the left. (B) 48-R9, 48-R11, or 48-R12 cells were serum starved and either left untreated or stimulated with 10 nM insulin for 1 to 60 min. Cells were collected in hot 3× Laemmli sample buffer, and lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific for phosphorylated and activated PKB/Akt (Phos-Akt) and then stripped and reprobed for PKB/Akt. Representative immunoblots from two independent experiments for each of the TC48-reconstituted clones are shown. Also shown are the respective phospho-Akt/Akt quantitations relative to the kinetics of PKB/Akt phosphorylation in TCPTP^−/− cells (as shown in Fig. 8, n = 3).