Shaozhong Dong

Tyrosine phosphorylation inhibits PKM2 to promote the Warburg effect and tumor growth

Tyrosine phosphorylation of pyruvate kinase M2 gives tumor cells a metabolic advantage. A Malignant Metabolic Switch Cancer cells show aberrant metabolism, consuming more glucose than do healthy cells and producing lactate even in the presence of abundant oxygen, rather than shifting to oxidative phosphorylation. This phenomenon is called the Warburg effect, after Otto Warburg, who described it many years ago. Building on recent research implicating inhibition of the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2) by phosphotyrosine binding as critical to the Warburg effect—and tumorigenesis—Hitosugi et al. explored the role of signaling from oncogenic forms of the fibroblast growth factor receptor type 1 (FGFR1) in mediating this metabolic switch. They found that FGFR1, a receptor tyrosine kinase, phosphorylated a tyrosine residue (Y105) on PKM2 itself. Further analysis revealed that this tyrosine residue was commonly phosphorylated in human cancers and that a mutant form of PKM2 lacking this tyrosine residue inhibited both “Warburg metabolism” and tumor growth. They thus propose that phosphorylation of PKM2 by oncogenic tyrosine kinases provides the very phosphotyrosine that binds to and inhibits PKM2 to induce the Warburg effect and promote tumor growth. The Warburg effect describes a pro-oncogenic metabolism switch such that cancer cells take up more glucose than normal tissue and favor incomplete oxidation of glucose even in the presence of oxygen. To better understand how tyrosine kinase signaling, which is commonly increased in tumors, regulates the Warburg effect, we performed phosphoproteomic studies. We found that oncogenic forms of fibroblast growth factor receptor type 1 inhibit the pyruvate kinase M2 (PKM2) isoform by direct phosphorylation of PKM2 tyrosine residue 105 (Y105). This inhibits the formation of active, tetrameric PKM2 by disrupting binding of the PKM2 cofactor fructose-1,6-bisphosphate. Furthermore, we found that phosphorylation of PKM2 Y105 is common in human cancers. The presence of a PKM2 mutant in which phenylalanine is substituted for Y105 (Y105F) in cancer cells leads to decreased cell proliferation under hypoxic conditions, increased oxidative phosphorylation with reduced lactate production, and reduced tumor growth in xenografts in nude mice. Our findings suggest that tyrosine phosphorylation regulates PKM2 to provide a metabolic advantage to tumor cells, thereby promoting tumor growth.

Sumoylation of nucleophosmin/B23 regulates its subcellular localization, mediating cell proliferation and survival

Nucleophosmin/B23 is a major multifunctional nucleolar phosphoprotein that plays a critical role in ribosome biogenesis and cell proliferation. Arf tumor suppressor binds B23 and enhances its sumoylation. However, the biological effects of this event remain unknown. Here we show that B23 is sumoylated on both Lysine 230 and 263 residues, but the latter is the major one. Mutation of K263, but not K230, into R abolishes its centrosomal and nucleolar residency. Moreover, Rb binds to wild-type B23, but fails to interact with K263R. Sumoylation enhances B23 binding to Rb. Consequently, B23 potently stimulates E2F1-mediated transcriptional activity, which is abolished in B23 K263R. Further, K263R mutation makes B23 vulnerable to caspase-3 cleavage and sensitizes cells to apoptosis. Surprisingly, K230R mutant strongly binds to phosphatidylinositol-3,4,5-trisphosphate and suppresses DNA fragmentation. Thus, B23 sumoylation regulates its subcellular localization, cell proliferation, and survival activities.

SRPK2 Promotes Leukemia Cell Proliferation by Phosphorylating Acinus and Regulating Cyclin A1

Serine/arginine (SR) protein-specific kinase (SRPK), a family of cell cycle-regulated protein kinases, phosphorylate SR domain-containing proteins in nuclear speckles and mediate the pre-mRNA splicing. However, the physiologic roles of this event in cell cycle are incompletely understood. Here, we show that SRPK2 binds and phosphorylates acinus, an SR protein essential for RNA splicing, and redistributes it from the nuclear speckles to the nucleoplasm, resulting in cyclin A1 but not A2 up-regulation. Acinus S422D, an SRPK2 phosphorylation mimetic, enhances cyclin A1 transcription, whereas acinus S422A, an unphosphorylatable mutant, blocks the stimulatory effect of SRPK2. Ablation of acinus or SRPK2 abrogates cyclin A1 expression in leukemia cells and arrest cells at G(1) phase. Overexpression of acinus or SRPK2 increases leukemia cell proliferation. Furthermore, both SRPK2 and acinus are overexpressed in some human acute myelogenous leukemia patients and correlate with elevated cyclin A1 expression levels, fitting with the oncogenic activity of cyclin A1 in leukemia. Thus, our findings establish a molecular mechanism by which SR splicing machinery regulates cell cycle and contributes to leukemia tumorigenesis.

Fibroblast Growth Factor Receptor 3 Associates with and Tyrosine Phosphorylates p90 RSK2, Leading to RSK2 Activation That Mediates Hematopoietic Transformation

ABSTRACT Dysregulation of the receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) plays a pathogenic role in a number of human hematopoietic malignancies and solid tumors. These include t(4;14) multiple myeloma associated with ectopic expression of FGFR3 and t(4;12)(p16;p13) acute myeloid leukemia associated with expression of a constitutively activated fusion tyrosine kinase, TEL-FGFR3. We recently reported that FGFR3 directly tyrosine phosphorylates RSK2 at Y529, which consequently regulates RSK2 activation. Here we identified Y707 as an additional tyrosine in RSK2 that is phosphorylated by FGFR3. Phosphorylation at Y707 contributes to RSK2 activation, through a putative disruption of the autoinhibitory αL-helix on the C terminus of RSK2, unlike Y529 phosphorylation, which facilitates ERK binding. Moreover, we found that FGFR3 interacts with RSK2 through residue W332 in the linker region of RSK2 and that this association is required for FGFR3-dependent phosphorylation of RSK2 at Y529 and Y707, as well as the subsequent RSK2 activation. Furthermore, in a murine bone marrow transplant assay, genetic deficiency in RSK2 resulted in a significantly delayed and attenuated myeloproliferative syndrome induced by TEL-FGFR3 as compared with wild-type cells, suggesting a critical role of RSK2 in FGFR3-induced hematopoietic transformation. Our current and previous findings represent a paradigm for tyrosine phosphorylation-dependent regulation of serine-threonine kinases.

Targeting 14-3-3 sensitizes native and mutant BCR-ABL to inhibition with U0126, rapamycin and Bcl-2 inhibitor GX15-070.

Small molecule tyrosine kinase inhibitors, such as imatinib, are effective therapies for BCR-ABL-mediated human leukemias. However, clinical drug resistance occurs, which warrants development of alternative and/or complementary therapeutic strategies to target critical downstream signaling molecules. We recently demonstrated that disrupting 14-3-3/ligand association by a peptide-based 14-3-3 competitive antagonist R18 induces significant apoptosis, partially through reactivation of AKT-inhibited proapoptotic FOXO3a, in FGFR1 fusion-transformed hematopoietic cells. Here, we report that targeting 14-3-3 by R18 effectively induced significant apoptosis in Ba/F3 and K562 cells expressing BCR-ABL, similarly through liberation and reactivation of FOXO3a. Moreover, R18 sensitized BCR-ABL-transformed cells to inhibition with MEK1 inhibitor U0126, Bcl-2 inhibitor GX15-070, or mTOR inhibitor rapamycin. Treatment with these reagents potentiated R18-induced reactivation of proapoptotic FOXO3a with enhanced expression of downstream transcription targets p27kip1 and Bim1. Furthermore, R18-induced apoptotic cell death in cells expressing diverse imatinib-resistant BCR-ABL mutants, including T315I. This inhibition was enhanced by R18 in combination with U0126 and rapamycin. Thus, our findings suggest that targeting 14-3-3 may potentiate the effects of conventional therapy for BCR-ABL-associated hematopoietic malignancies, and overcome drug resistance.

Epidermal Growth Factor Stimulates RSK2 Activation through Activation of the MEK/ERK Pathway and Src-dependent Tyrosine Phosphorylation of RSK2 at Tyr-529*

The Ser/Thr kinase ribosomal S6 kinase 2 (RSK2) has been demonstrated to phosphorylate transcription factor CREB (cyclic AMP-responsive-binding protein) and histone H3 in response to mitogenic stimulation by epidermal growth factor (EGF). EGF activates the MEK/ERK pathway to activate RSK2. We recently reported that receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) directly tyrosine phosphorylates RSK2 at Tyr-529, which consequently regulates RSK2 activation by facilitating inactive ERK binding to RSK2 that is required for ERK-dependent phosphorylation and activation of RSK2 (Kang, S., Dong, S., Gu, T. L., Guo, A., Cohen, M. S., Lonial, S., Khoury, H. J., Fabbro, D., Gilliland, D. G., Bergsagel, P. L., Taunton, J., Polakiewicz, R. D., and Chen, J. (2007) Cancer Cell 12, 201–214). Here we report that upon treatment of EGF, RSK2 was tyrosine-phosphorylated at Tyr-529 and activated in 293T and COS7 cells that do not express FGFR3. In contrast to FGFR3, the receptor tyrosine kinase EGF receptor did not directly phosphorylate RSK2 at Tyr-529 in an in vitro kinase assay using recombinant RSK2 and active EGF receptor or FGFR3. By mass spectroscopy-based studies, we identified Src tyrosine kinase family members Src and Fyn as upstream kinases of RSK2 Tyr-529. Treatment of Src inhibitor PP2 effectively attenuated EGF-dependent activation and Tyr-529 phosphorylation of RSK2, suggesting that Src family members are the kinases that phosphorylate RSK2 at Tyr-529 in response to EGF. Src and Fyn were able to directly phosphorylate RSK2 at Tyr-529 in the in vitro kinase assay. In vitro reconstitution of Tyr-529 phosphorylation by Src in glutathione S-transferase-tagged RSK2 enhanced inactive ERK binding to RSK2 wild type, but not the Y529F mutant. Together, our findings suggest that Src-dependent phosphorylation at Tyr-529 facilitates inactive ERK binding to RSK2, which might be a general requirement for RSK2 activation by EGF through the MEK/ERK pathway.