Dual-Specificity Phosphatases In

Dual-speciﬁcity phosphatases in NUCLEAR SIGNALING www.abcepta.com 2 2 1 1 1 S.Gramatikova PhD, K.Gramatiko PhD, J.Mountzouris PhD, T.Gilliam & C.Wu PhD

(1) Abcepta Inc., 10320 Camino Santa Fe, Ste G, San Diego, CA 92121 (2) Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037 ABGENT has hundreds of phosphorylation-related antibodies which cover key targets for cell cycle con- Dual-speciﬁcity phosphatases trol, development/differentiation, and signal transduction. Visit www.abgent.com for a complete listing.

Selected Abgent Products Cyclin D ? Examples for dual-specificity phosphatases and their biological functions CDC25A PCNA CDK4 Figure Target Tissue/Cell line Cat# A. B. Rb E2F Cyclin E A. DUSP4 Human breast carcinoma AP8447b Gene Name Role in nuclear signaling Cellular process / disease B. CDC25B Human breast carcinoma AP7256c CDK2 CDC25C + C. CDK1 Human breast carcinoma AP7517d CDC25A, B, C cell-division cycle 25 DNA damage Cell cycle control, checkpoint pathways E2F P P D. CDK6 Human breast carcinoma AP7522b Cyclin B Rb P CDC14A, B, C cell division cycle 14 p53 regulation Cell cycle control, cytokinesis, cancer C. D. G1 PDGFRA Human carcinoma (HeLa) AP7666j E. M CDC25A PTEN phosphatase and tensin homolog DNA repair Cell cycle control, chromosome stability F. DUSP6 Human carcinoma (HeLa) AT1830a + CDK1 G2 S PTPN11 SHP2 Transcriptional regulation Mitogenic activation, metabolic control G. Cyclin D1 Mouse lung tissue lysate AP2612d Cyclin A Cyclin A DUSP1 dual-specificity phosphatase 1 Transcriptional regulation Cell cycle control, immune response H. Cyclin E1 A2058 cell line lysate AP6270b CDK1 CDK2 I. CDC25A HeLa cell line lysate AP6272c DUSP2 dual-specificity phosphatase 2 Nuclear accumulation of ERK Immune response, heat shock J. CDK2 Jurkat cell line lysate AP7518b E. F. Wee1/Myt1 DUSP4 dual-specificity phosphatase 4 Nuclear accumulation of ERK Control of cell cycle and MAP kinases – Cyclin A K. CDK4 HL-60 cell line lysate AP7520b DUSP5 dual-specificity phosphatase 5 Nuclear translocation Immune response L. Cyclin B1 NCI-H460 cell line lysate AP7598a CDK2 DUSP6 dual-specificity phosphatase 6 FGF signaling to the nucleus Development, postnatal lethality M. DUSP3 SK-BR-3 cell line lysate AP8478a CDC25B DUSP7 dual-specificity phosphatase 7 FGF signaling to the nucleus Development DUSP9 dual-specificity phosphatase 9 Transcriptional regulation Development Lung A2058 HeLa Jurkat HL-60 NCI-H460 SK-BR-3 DUSP10 dual-specificity phosphatase 10 Transcriptional regulation Immune response 95 150 95 Fig. 1 CDC25 phosphatases promote mammalian cell-cycle progression. Dual- 95 72 specificity phosphatases (DSPases) have a central role in the complex regulation of signaling pathways that are 55 DUSP12 dual-specificity phosphatase 12 Heat stress response Cell survival, diabetes 95 75 37 55 55 involved in cell stress responses, proliferation and death (1). The cell-division cycle 25 (CDC25) family of DSPases 55 regulates cell-cycle progression by dephosphorylating and activating cyclin-dependent kinases (CDKs). In the 25 DUSP14 dual-specificity phosphatase 14 Transcriptional regulation Immune response, CD28 signaling 36 55 event of DNA damage, CDC25 are key targets of the checkpoint machinery that ensures genetic stability. Inactive 36 36 36 37 CDKs are phosphorylated at adjacent threonine and tyrosine residues near their amino termini. Dephosphoryla- DUSP22 dual-specificity phosphatase 22 STAT3 activation, ERα signaling Immune response, proliferation 28 28 28 tion at both sites by CDC25 phosphatases catalyses their activation and allows the CDKs to propagate cell-cycle 25 28 14 EPM2A laforin β-catenin accumulation in nucleus Lafora progressive myoclonus epilepsy 28 signal transduction (1-12). Indicated in red are the protein targets for ABGENTʼs antibody products.

G. H. I. J. K. L. M.

CDK1 CHK1, p38 CDK1 Ub Ub Overexpression of CDC25 in cancer SCF/βTRCP Ub Ub P P P P Ub P APC/C Ub S18 S76 S82 S88 S116 CDC25A NES DSGFCLDS KEN NLS 47% Y Gliomas S124 S178 S279 S293 T507 P P P P P 14-3-3 14-3-3

p38 CHK1 and/or CHK2 Thyroid 17-69% N 36-64% Y 41% N 57% Y Laryngeal CHK1 MELK, MK2 CHK1 p38 MELK, MK2 CHK1 14-3-3 14-3-3 14-3-3 P P P P P P S151 S169 S230 S249 S323 S563 CDC25B NES DDG NLS C S186/187 S353 S375 Breast 70% N 57% Y 46-66% Y 48-79% Esophageal P SCF/βTRCP Ub P P Ub Ub CK2 CDK1/Cyclin A Aurora A MK2

ERK1/2 78% Y Gastric Ub Hepatocellular 56% Y 20% N Ub P Ub CDC25C NES NLS S191 S198 S214 S216 T236 P P P P P 73% Y 13% ? Endometrial 14-3-3 Ovarian 30% Y 30% Y A PLK1/PLK3 CDK1 C-TAK1, CHKs CK2

30% Y Prostate or or T T ec t ec t V W S292A V W T506A p-S292 p-T506 Colorectal 47-53% Y 43-67% Y 27% ? (AP3048a) (AP3051a)

CDC25A CDC25A B C Non-Hodgkin lymphoma 50% Y 65% Y 15% Y Fig. 2 Overexpression of CDC25 protein in human cancers. CDC25 proteins are overexpressed in a wide variety of CDC25A, B or C cancers. Percentages of tumors in which CDC25A, CDC25B or CDC25C are overexpressed are indicated. CDC25 overexpression is linked to A B C clinicopathological features, including tumor grade or stage, metastases, depth of invasion, residual or recurrent disease. Cases for which overexpression (%) several studies reported contradictory prognostic values are marked by a (C), and studies in which clinicopathological features were not assessed are indicated by an (?). The intensity of the red circles in the human body corresponds to the cancer mortality (2, 5).

Prognostic value? Yes (Y), No (N), Fig. 3 Domain organization of CDC25 homologs (A) and detection of CDC25 in transfected cells (B, C). ABGENTʼs antibody #AP3048a was generated against synthetic phosphopeptide corresponding to amino acids surrounding S292 of hu- Contradictory (C), Not examined (?) man CDC25A (B). The antibody was used in Western blot to detect Phospho-CDC25A-S292 in cells transfected with wild type (wt) or mutant S292A of CDC25A. Antibody #AP3051a was generated against phosphopeptide corresponding to amino acids surrounding T506 of human Fig.2 CDC25A (C). The antibody was used to detect Phospho-CDC25A-T506 in cells transfected with wild type or mutant T506A of CDC25A.

Control of the cell cycle Product abbreviations C-phase DUSP4: dual specificity phosphatase 4; MAP kinase phosphatase 2; VH1 homologous phosphatase 2 CDC25A & B: cell division cycle 25 homolog A & B CDK1: cell division cycle 2, G1 to S and G2 to M; cyclin-dependent kinase 1; p34 protein kinase; CDC2 CDK6: cyclin-dependent kinase 6; cell division protein kinase 6 G1 S G2 P PM M A T PDGFRA: platelet-derived growth factor receptor, alpha polypeptide DUSP6: dual specificity phosphatase 6; MAP kinase phosphatase 3 P P P Cyclin D1: B-cell CLL/lymphoma 1; G1/S-specific cyclin D1; CCND1 CDH1 APC/C CDH1 APC/C APC/C APC/C APC/C P P Cyclin E1: cyclin Es; cyclin Et; CCNE1 CDH1 CDH1 CDH1 P CDC20 CDH1 CDK2: cyclin-dependent kinase 2; cdc2-related protein kinase; cell devision kinase 2; p33 protein kinase

s CDK4: cyclin-dependent kinase 4; cell division kinase 4; melanoma cutaneous malignant, 3 e

s EMI1 EMI1 : G2/mitotic-speciﬁc cyclin B1; CCNB1 a CCNB1 e DUSP3: dual speciﬁcity phosphatase 3; vaccinia virus phosphatase VH1-related; VHR

ncr SKP2

ty i SKP2 β-TrCP SKP2 β-TrCP SKP2 EMI1 SCF SCF v i SCF SCF SCF SCF

ct i References

n a β-TrCP 1. Patterson KI, Brummer T, OʼBrien PM and Daly RJ. (2009) Dual-speciﬁcity phosphatases: critical regulators with diverse cellular cl i P P targets. Biochem J. 418(3), pp.475-489. p21 p27 p21 p27 p21 p27 CDC 25A SCF p21 CDC 25A p21 p27 CDC 25A -cy CDC 25A WEE1 P P 2. Boutros R, Lobjois V and Ducommun B. (2007) CDC25 phosphatases in cancer cells: key players? Good targets?. Nat Rev Cancer. P 7(7), pp.495-507. CDK 3. Jeffrey KL, Camps M, Rommel C and Mackay CR. (2007) Targeting dual speciﬁcity phosphatases: manipulating MAP kinase signalling and immune responses. Nat Rev Drug Discov. 6(5), pp.391-403. CDK1 Cyclin A CDK1 CDK1 Cyclin A CDK1 Cyclin A Cyclin A 4. Frescas D and Pagano M. (2008) Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP: tipping the scales of cancer. CDK2 Cyclin B CDK2 CDK2 Cyclin B CDK2 Cyclin B Nat Rev Cancer. 8(6), pp.438-449. 5. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T and Thun MJ. (2008) Cancer statistics, 2008. CA Cancer J Clin. 58(2), pp.71- 96. APC/C activity 6. Brezak MC, Kasprzyk PG, Galcera MO, Lavergne O and Prévost GP. (2008) CDC25 inhibitors as anticancer agents are moving forward. Anticancer Agents Med Chem. 8(8):857-62. CDK–cyclin activity 7. Planchon SM, Waite KA and Eng C. (2008) The nuclear affairs of PTEN. J Cell Sci. 121(Pt 3), pp.249-253.

8. Lyon MA, Ducruet AP, Wipf P and Lazo JS. (2002)Dual-specificity phosphatases as targets for antineoplastic agents. Nat Rev Drug Discov. 1(12), pp.961-976. Fig.4 Control of the cell cycle by the ubiquitin–proteasome system. The cell division cycle is regulated primarily by the activity of cyclindependent kinases (CDKs) and protein degradation by the ubiquitin–proteasome system (UPS). Each CDK complex contains one of many activating subunits, termed cyclins, the levels of which oscillate during the cell cycle. CKIs (CDK inhibitors), such as p27 and p21, inhibit CDK activity and promote cell cycle arrest and/or delay. SCF complexes and the APC/C (anaphase-promoting complex/cyclosome) provide the specific, rapid and timely proteolysis of cell 9. Dickinson RJ and Keyse SM. (2006)Diverse physiological functions for dual-specificity MAP kinase phosphatases. J Cell Sci. cycle regulators, which ultimately controls CDK1 and CDK2 to finely modulate their activities during cell cycle progression. The best characterized cell cycle ubiquitin ligases are SCFSKP2, SCFFBXW7 (not shown), SCFβ-TrCP, APC/CCDH1 and APC/CCDC20. SCFSKP2 is a positive regulator of cell cycle progression 119(Pt 22), pp.4607-4615. (by promoting the degradation of p21 and p27), whereas SCFβ-TrCP is both a positive and negative regulator of the cell cycle (by targeting CDC25A (cell division cycle 25A), claspin, WEE1 and EMI1 (also known as F-box protein 5)). APC/CCDH1 and APC/CCDC20 always attenuate CDK1 activity (by directing the degradation of cyclins A and B), except in early mitosis, when APC/CCDC20 targets p21 for degradation. Finally, SCFFBXW7 attenuates CDK1 and CDK2 by inducing the degradation of cyclin E. SCF complexes and the APC/C control each other, with SKP2 being ubiquitylated by APC/CCDH1 in G1 and SCFβ-TrCP 10. Lang R, Hammer M and Mages J. (2006) DUSP meet immunology: dual specificity MAPK phosphatases in control of the inflam- targeting EMI1, which is an inhibitor of APC/CCDH1, for proteolysis in early mitosis. Additionally, SCF complexes and the APC/C share common substrates that are targeted by their respective ubiquitin ligase(s) only at particular times during the cell cycle. For example, SCFSKP2 targets p21 for degradation at matory response. J Immunol. 177(11), pp.7497-7504. G1–S, whereas APC/CCDC20 targets p21 during prometaphase. This scenario is also true for the targeted degradation of CDC25A by APC/CCDH1 in G1 phase, which is followed by SCFβ-TrCP-mediated degradation during S phase. Moreover, phosphorylation by CDKs modulates the activity of SCF complexes and the APC/C. CDK activity inhibits binding of CDH1 to the APC/C while promoting the activation of APC/CCDC20, and phosphorylation of certain SCF substrates by CDKs allows recognition by the F-box protein subunit. β-TrCP, β-transducin repeat-containing protein; CDH1, also known as FZR1 (fizzy/cell division 11. Hutchins JR and Clarke PR. (2004) Many fingers on the mitotic trigger: post-translational regulation of the Cdc25C phos- cycle 20 related 1); FBXW7, F-box protein with WD domain 7; SKP2, S-phase kinase-associated protein 2 (4). phatase. Cell Cycle. 3(1), pp.41-45. 12. Wu TR, Hong YK, Wang XD, Ling MY, Dragoi AM, Chung AS, Campbell AG, Han ZY, Feng GS and Chin YE. (2002) SHP-2 is a dual-specificity phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues in nuclei. J Biol Chem. 277(49), pp.47572-47580.