pubmed.ncbi.nlm.nih.gov

Reciprocal regulation of protein kinase and pyruvate kinase activities of pyruvate kinase M2 by growth signals - PubMed

  • ️Tue Jan 01 2013

Reciprocal regulation of protein kinase and pyruvate kinase activities of pyruvate kinase M2 by growth signals

Xueliang Gao et al. J Biol Chem. 2013.

Abstract

Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2 interacts with tyrosine phosphopeptide, and the interaction with the tyrosine phosphopeptide affects the pyruvate kinase activity of PKM2. Our experiments suggest that PKM2 is also an active protein kinase (Gao, X., Wang, H., Yang, J. J., Liu, X., and Liu, Z. R. (2012) Mol. Cell 45, 598-609). We report here that growth signals reciprocally regulate the pyruvate kinase and protein kinase activities of PKM2 by different mechanisms. On the one hand, growth signals induce protein tyrosine phosphorylations. The tyrosine-phosphorylated protein(s) regulates the conversion of pyruvate kinase and protein kinase of PKM2 by directly interacting with PKM2. Binding of the tyrosyl-phosphorylated proteins at the fructose 1,6-bisphosphate-binding site converts the tetrameric PKM2 to a dimer. On the other hand, growth stimulations also lead to PKM2 phosphorylation, which consequently regulates the conversion of protein kinase and pyruvate kinase activities. Growth factor stimulations significantly increase the dimer/tetramer PKM2 ratio in cells and consequently activate the protein kinase activity of PKM2. Our study suggests that the conversion between the pyruvate kinase and protein kinase activities of PKM2 may be an important mechanism mediating the effects of growth signals in promoting cell proliferation.

Keywords: Bioenergetics; Growth Factors; Protein Kinases; Pyruvate Kinase; STAT3.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.

Tyrosine-phosphorylated p68 interacts with PKM2. A, left panel, GelCode staining SDS-PAGE analyses of proteins pulled down from nuclear extracts of T98G cells by different peptides coupled to agarose beads. The peptides used are as follows: PepY593, PhosPepY593, D-PhosPepY593, and PhosPepY567. The arrow indicates the protein band that was cut out and subjected to trypsin digestion followed by MALDI-TOF/TOF analyses. M is molecular weight markers. Right panel, amino acid sequence of PKM2. The boldface and underlined sequences indicate the peptide fragments identified by MALDI-TOF/TOF. B, interaction of the peptides, the PepY593 and D-PhosPepY593, with rPKM2. The recombinant PKM2 pulled down (PD) by the peptides was examined by immunoblot (IB) using antibody against PKM2 (IB:PKM2). Pulldown by rPKM2 is a control without peptide pulldown. C, pulldown (PD) of p68 from nuclear extracts (NE) of T98G cells by rPKM2 was examined by immunoblot using antibody p68-RGG (IB:p68). The tyrosine phosphorylation of p68 was indicated by strip-blot of the pulldown using antibody against phosphotyrosine (IB:PY20). D, co-immunoprecipitations of exogenously expressed HA-p68s (WT and Y593F, Vec is vector alone) with endogenous PKM2 from nuclear extracts of T98G cells were examined by immunoblot with anti-HA (IP:PKM2, IB:HA). IBs of the nuclear extracts using anti-HA indicate the expression of HA-p68 (IB:HA). IBs of histone 2A (IB:H2A) are the loading controls. E, top panel, co-immunoprecipitations of p68 with endogenous PKM2 from nuclear and cytoplasmic (Cyto) extracts of T98G cells were examined by immunoblot with antibody p68-RGG (IP:PKM2, IB:p68). Bottom panel, co-immunoprecipitations of p68 with exogenously expressed HA-PKM2 from nuclear and cytoplasmic extracts of T98G cells were examined by immunoblot with antibody p68-RGG (IP:HA, IB:p68). IBs of HAPKM2 indicate the immunoprecipitated HA-PKM2 from the extracts. IBs of GAPDH (IB:GAPDH) and lamin A/C (IB:Lamin A/C) are the loading controls. Beads in B and C are the control pulldown using agarose beads alone.

FIGURE 2.
FIGURE 2.

PKM2 interacts with tyrosyl phosphopeptide. A, Trp fluorescence emission spectrum of the rPKM2 (4 μ

m

) with the titration of various different concentrations (indicated) of peptide

d

-PhosPepY593. Binding of the peptide to the rPKM2 was indicated by the decrease in fluorescence intensity at 334 nm. B, Trp fluorescence emission intensity of the rPKM2 at 334 nm in the presence of various peptides (2 μ

m

) D-PhosPepY593 (red circles), PepY593 (blue crosses), PhosPepY567 (green diamonds), and D-PhosPepY593 + 2 m

m

FBP (black squares). The y axis is the fluorescence intensity at 334 nm. The x axis is the peptide concentration. C, dissociation constants (Kd) of the rPKM2 with the peptides D-PhosPepY593, PepY593, and PhosPepY567 were determined by the data fitting to Langmuir equation (assume 1:1 binding). The Kd values were determined based on data fitting on three independent measurements.

FIGURE 3.
FIGURE 3.

A, co-immunoprecipitation of p68 (IP:p68) with exogenously expressed HA-PKM2 in cellular extracts of T98G cells supplemented with various concentrations of FBP was examined by immunoblot using the anti-HA antibody (IB:HA). B, pulldown of the rPKM2 by the HA-p68 immunopurified from T98G cellular extracts (PD:HA-p68) was analyzed by immunoblot using antibody against PKM2 (IB:PKM2). The pulldown experiments were performed in the presence of various concentrations of FBP (indicated in figure). IB:p68 indicates amount of HA-p68 used in the pulldown. C, co-immunoprecipitation of p68 with the exogenously expressed HA-PKM2 and mutants (indicated) in the cellular extracts prepared from T98G cells. Top panel, antibody p68-RGG was used as immunoprecipitation antibody (IP:p68). Co-immunoprecipitations of HA-PKM2 and mutants with p68 were examined by immunoblot using antibody against PKM2. Immunoblot of p68 (IB:p68) indicated amounts of p68 that were immunoprecipitated down. Bottom panel, anti-HA antibody was used as immunoprecipitation antibody (IP:HA). Co-immunoprecipitations of p68 with HA-PKM2 and mutants were examined by immunoblot using antibody against p68 (IB:p68). D, size exclusion chromatography fractionation of the wild type rPKM2 in the presence of different peptides (3 μ

m

) D-PhosPepY593 (upper panel), PepY593 (middle panel), and PhosPepY567 (lower panel). 100 μl of the rPKM2 in 15 μ

m

was loaded onto the column. The chromatography fractions F10–F18 were collected using a FPLC 300 μl per fraction. The collected fractions were immunoblotted using the antibody PabPKM2 (IB:PKM2). IgG HC in A–C are the Ponceau S stain of antibody heavy chain, representing the amounts of antibody used in the immunopurifications.

FIGURE 4.
FIGURE 4.

Tyrosine phosphoprotein/peptide reciprocally regulates protein kinase and pyruvate kinase activities of PKM2. A and B, phosphorylation of GST-Stat3 by the rPKM2 (10 μg/ml) and the R399E (10 μg/ml) (A) or by R399E (10 μg/ml) (B) in the presence of different peptides (3 μ

m

) D-PhosPepY593, PepY593, and PhosPepY567 was revealed by immunoblot assays using antibody against Tyr-705-phosphorylated Stat3 (IB:pY-Stat3). C, phosphorylation of GST-Stat3 by the rPKM2 (10 μg/ml) or the rPKM1 (10 μg/ml) in the presence of HA-p68 that was immunopurified from nuclear extracts of T98G cells and 5 m

m

of PEP was revealed by immunoblot assays using antibody against Tyr-705 phosphorylated Stat3 (IB:pY-Stat3). In some phosphorylation reactions, 5 m

m

FBP or 5 m

m

ADP was added to the reaction. Immunoblots of Stat3 (IB:Stat3), HA-p68 (IB:HA), and PKM2 (IB:PKM2) indicate the amounts of GST-Stat3, HA-p68, and the rPKM2 used in each phosphorylation reaction. D, phosphorylation of Stat3 by immunopurified HA-PKM2 from nuclear extracts (HAPK) of T98G cells in the presence of ATP or PEP was analyzed by immunoblot using the antibody against the Tyr-705-phosphorylated Stat3 (IB:pY-Stat3). p68 was knocked down by duplex RNAi. Immunoblot of Stat3 (IB:Stat3) shows the amounts of Stat3 that were immunoprecipitated from the nuclear extracts. Immunoblot of HA (IB:HA) indicates the expression levels of HA-PKM2. Ponceau S stain of IgG heavy chain (IgG:HC) indicates the amounts of antibody used in each PKM2 purification from nuclear extracts. E, pyruvate kinase activity of the rPKM2 (5 μg/ml) in the presence of different peptides (2 μ

m

) D-PhosPepY593, PepY593, and PhosPepY567 or buffer was analyzed by the method described by Christofk et al. (9). The pyruvate kinase activity of the rPKM2 was expressed as relative pyruvate kinase activity by defining the activity in the presence of buffer as 100. The error bars represent the standard deviations of four measurements.

FIGURE 5.
FIGURE 5.

A, top panels, phosphorylation of nuclear Stat3 was analyzed by immunoblot using antibody against Tyr-705- phosphorylated Stat3 (IB:pY-Stat3) in the nuclear extracts prepared from different cells. Immunoblot of Stat3 (IB:Stat3) indicates the Stat3 levels in the nuclear extracts. Immunoblot of lamin (IB:Lamin A/C) is a loading control. Bottom panels, phosphorylation of p68 in nuclear extracts was analyzed by immunoblot of the immunoprecipitated p68 (IP:p68) using the antibody Tyr(P)-100 (IB:pY-100). Immunoblot of p68 (IB:p68) indicates the amounts of p68 that were immunoprecipitated from nuclear extracts (IB:p68). B, quantitation of phosphorylation levels of Stat3 (upper panel) and the immunoprecipitated p68 (bottom panel) in nuclear extracts of different cells. The phosphorylation levels of Stat3 and immunoprecipitated p68 were presented as relative phosphorylation levels (pY-Stat3/Stat3) and (pYp68/p68), respectively. The error bars are standard deviations from four independent experiments. C, interaction of GST-Stat3, the bacterially expressed rPKM2, and the HA-p68 immunopurified from T98G cells was analyzed by GST-pulldown using the glutathione beads. The GST-Stat3 (2 μg/ml), the rPKM2 (2 μg/ml), and the HA-p68 were incubated in 500 μl at room temperature. The GST-Stat3 was precipitated by the GST beads. Co-precipitations of the HA-p68 and the rPKM2 from the mixture were analyzed by immunoblot using antibodies against p68 (IB:p68) and PKM2 (IB:PKM2). The strip blot using anti-phosphotyrosine indicates the tyrosine phosphorylation of the precipitated HA-p68. Immunoblot of Stat3 (IB:Stat3) indicates the amounts of GST-Stat3 that were precipitated by GST beads. D, co-immunoprecipitations of PKM2 and Stat3 with HA-p68 in the cellular extracts of T98G cells. The exogenously expressed HA-p68 was immunoprecipitated using anti-HA antibody. The amounts of p68 precipitated from the extracts were examined by immunoblot using antibody against p68. The co-precipitation of PKM2 and Stat3 was probed by immunblot using antibodies against PKM2 (IB:PKM2) and against Stat3 (IB:Stat3). IgG HC in A and D are the Ponceau S stain of antibody heavy chain, representing the amounts of antibody used in immunoprecipitation of HA-p68 from the extracts.

FIGURE 6.
FIGURE 6.

Growth stimulation activates protein kinase activity of PKM2. A, size exclusion chromatography fractionation of cell lysates prepared from SW480 cells that were treated/untreated by 25 ng/ml EGF for 8 h. The cell lysates (100 μl) were loaded onto the column. The chromatography fractions F9–F20 were collected using an FPLC at 300 μl per fraction. The collected fractions were immunoblotted using the antibody against PKM2 (IB:PKM2). B, PKM2 levels in the nuclear extracts (NE) of SW480 cells that were treated with EGF (25 ng/ml) at different time points (indicated) were examined by immunoblot of the nuclear extracts using antibody against PKM2 (IB:PKM2). Immunoblot analyses of PKM2 (IB:PKM2) in the whole cell lysate (WCL) indicate the cellular levels of PKM2. C, left panel, tyrosine phosphorylation of p68, co-precipitation of PKM2 with p68, and phosphorylation of Stat3 in the nuclear extracts of SW480 cells that were treated with EGF (25 ng/ml) at different time points (indicated) were examined by immunoblot analyses of immunoprecipitates (IP:68) using the anti-phosphotyrosine antibody (IB:pY20), anti-PKM2 antibody (IB:PKM2), and anti-phospho-state3 (IB:pY-Stat3). Immunoblot analyses of p68 (IB:p68) in the whole cell lysate indicate the cellular levels of PKM2. Right panel, pyruvate kinase activity of extracts prepared from the EGF-treated SW480 cells. The pyruvate kinase activity is presented as relative pyruvate kinase activity (Relative PK) by defining untreated SW480 cells as 100. Immunoblot of β-actin (IB:β-actin) and lamin A/C (IB:Lamin A/C) in A and C are the loading controls.

FIGURE 7.
FIGURE 7.

A, phosphorylations of GST-Stat3 by PKM2 immunopurified from extracts of Ba/F3 cells using antibodies against PKM2 (αPK) or Tyr-105-phosphorylated PKM2 (αpY105) were analyzed by immunoblot using antibody against Tyr-705-phosphorylated Stat3 (IB:pY-Stat3). IB:PKM2 indicates the amount of PKM2 that immunoprecipitated from the extracts. IgG HC is the IgG heavy chain indicating the amount of antibody used in the immunoprecipitation. B, phosphorylations of GST-Stat3 by the rPKM2 (10 μg/ml), rR399E (10 μg/ml), or rY105E (10 μg/ml) in the presence of 2 m

m

PEP were analyzed by immunoblot using antibody against Tyr-705-phosphorylated Stat3 (IB:pY-Stat3). CBB is Coomassie Brilliant Blue stain of GST-Stat3 indicating amount of GST-Stat3 used in each phosphorylation reaction. C, in vitro phosphorylations of GST-Stat3 by recombinant Y105E and Y105F PKM2 mutants in the presence and absence of phosphorylated HA-p68 immunopurified from T98G cells were analyzed by immunoblot using antibody against Tyr-705 phosphorylated Stat3 (IB:pY-Stat3). IB:HA indicates the amount of HA-p68 used in each phosphorylation reaction. IB:Stat3 indicates the amount of GST-Stat3 used in each phosphorylation reaction. D, hypothetic model that illustrates the functional role of growth signals in the regulation of protein kinase and pyruvate kinase activities of PKM2.

Similar articles

Cited by

References

    1. Elbers J. R., van Unnik J. A., Rijksen G., van Oirschot B. A., Roholl P. J., Oosting J., Staal G. E. (1991) Pyruvate kinase activity and isozyme composition in normal fibrous tissue and fibroblastic proliferations. Cancer 67, 2552–2559 - PubMed
    1. Hacker H. J., Steinberg P., Bannasch P. (1998) Pyruvate kinase isoenzyme shift from L-type to M2-type is a late event in hepatocarcinogenesis induced in rats by a choline-deficient/dl-ethionine-supplemented diet. Carcinogenesis 19, 99–107 - PubMed
    1. Dombrauckas J. D., Santarsiero B. D., Mesecar A. D. (2005) Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis. Biochemistry 44, 9417–9429 - PubMed
    1. Zwerschke W., Mazurek S., Massimi P., Banks L., Eigenbrodt E., Jansen-Dürr P. (1999) Modulation of type M2 pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein. Proc. Natl. Acad. Sci. U.S.A. 96, 1291–1296 - PMC - PubMed
    1. Altenberg B., Greulich K. O. (2004) Genes of glycolysis are ubiquitously overexpressed in 24 cancer classes. Genomics 84, 1014–1020 - PubMed

Publication types

MeSH terms

Substances