Crystal Structure And Substrate Specificity Of PTPN12

PTPN12,which is encoded by the PTPN12 gene and was initially cloned in 1992,is a member of the non-receptor protein tyrosine phosphatase subfamily.PTPN12 contains 780 amino acids,has a molecular weight of 112 kDa,and is expressed ubiquitously in a wide variety of tissues and cell types.PTPN12 is located mainly in the cytoplasm and is highly expressed in the hematopoietic system,particularly in the thymus,spleen,and liver,but it is also found in the brain and heart.PTPN12 consists of an NH2-terminal catalytic domain and a long COOH-terminal domain.The large noncatalytic C-terminal region is rich in PEST sequences,which are found in many rapidly degrading proteins.PTPN12,however,appears to be quite stable.The main function of the noncatalytic segment of PTPN12 is to mediate the interactions of PTPN12 with its substrates and/or adaptor proteins.PTPN12 plays an essential function in early embryogenesis,and ablation of the PTPN12 gene leads to early embryonic lethality,correlating well with the high expression of the protein during embryonic development..Analysis of PTPN12 null mutant embryos revealed defects in the embryonic mesenchyme and subsequent defects in vascularization,liver development,and somatogenesis.PTPN12 plays important roles in various physiological processes,such as embryonic development,cell migration,immune response and osteoclast differentiation.PTPN12 is recently reported to be a tumor suppressor in triple negative breast cancer(TNBC),through its capacity to dephosphorylate oncogenic receptor protein tyrosine kinases(PTKs),such as EGFR,HER2 and PDGFR.PTPN12 is frequently compromised in breast cancer by deletion,inactivating sequence variants,or loss of expression.Loss of PTPN12 phosphatase activity leads to aberrant acinar morphogenesis and cellular transformation in mammary epithelial cells.Importantly,in breast cancer cells exhibiting PTPN12 deficiency,restoring PTPN12 expression to levels observed in normal mammary epithelial cells suppresses proliferation,tumorigenesis,and metastasis.A mouse model of ErbB2-dependent breast cancer using a conditional PTPN 12-deficient mouse showed that lack of PTPN12 in breast epithelial cells accelerated breast cancer development and lung metastases in vivo.Nevertheless,the molecular basis underlying the substrate specificity of PTPN 12 remains uncertain.To visualize the molecular architecture of PTPN 12,diffraction-quality crystals corresponding to the catalytic domain of a somatic cancer mutant(K61R)of PTPN 12 have been obtained with 2.4A resolution.Here,enzymological and crystallographic studies have enabled us to identify two distinct structural features that are crucial determinants of PTPN 12 substrate specificity:the pY+1 site binding pocket and specific basic charged residues along its surface loops.Key structurally plastic regions and specific residues in PTPN 12 enabled recognition of different HER2 phosphorylation sites and regulated specific PTPN 12 functions.In addition,the structure of PTPN 12 revealed a CDK2 phosphorylation site in a specific PTPN 12 loop.Taken together,our results not only provide the working mechanisms of PTPN 12 for desphosphorylation of its substrates but will also help in designing specific inhibitors of PTPN 12.Research purposesTo identify the crystal structure of PTPN 12 by crystallographic studies,to investigate the molecular basis underlying the substrate specificity of PTPN 12 by crystallographic and enzymological studies,to explore the impact of the substrate specificity of PTPN 12 on breast cancer cellular functions by cytological studies.Research methods1)Construction of plasmidsDNA-encoding residues 1-305 of human PTPN 12 catalytic domain was subcloned into pET-15b expression vector with an N-terminal His tag.All of the mutations were generated with the Quikchange kit from Stratagene.DNA-encoding full length and catalytic domain of human PTPN 12 were subcloned into pcDNA3.1 expression vector with an N-terminal Flag tag seperately.DNA-encoding full length of human CDK2 was subcloned into pGEX-6P-2 expression vector with an N-terminal GST tag.2)Protein expression and purificationThe His-tagged PTPN12 wide type and mutant catalytic domain proteins were expressed in BL21-DE3 Escherichia coli.By Ni-NTA Agrose and Superdex 75,the His-PTPN12 Catalytic Domain proteins of purity over 95%were obtained.The GST-tagged CDK2 protein were expressed in BL21-DE3 Escherichia coli.By GST-beads,the GST-CDK2 protein of purity over 90%was obtained.3)Crystallisation and structure determinationBy hanging drop vapor diffusion method of protein crystallization,the crystals with cubic shape emerged.The data were collected to 2.4A resolution using 0.98A X-ray wave length and analyzed by HKL2000.4)Assays of phosphatase activityAll phosphatase assays were carried out in 3,3-dimethylglutaric acid(DMG)buffer(pH 7.0,3,3-dimethyl glutarate 50 mM,EDTA 1 mM,DTT 2 mM,and the ionic strength was adjusted to 0.15 M by NaCl addition)with 96-well plates at 37?.All reactions commenced by mixing enzyme with substrates at different concentrations.For pNPP,the reaction was terminated by mixing with NaOH and then measured at 405 nm absorbance.For DiFMUP,the reaction was measured by excitation at 358 nm and emission was detected at 455 nm.For OMFP,the reaction was measured by excitation at 485 nm and emission at 525 nm.The data were gathered and analyzed by GraphPad Prism5(GraphPad Software).The peptides were dissolved in water,and the concentration of peptides was determined by complete dephosphorylation and determination of the released inorganic phosphate.The phosphatase assay for phospho-peptides was carried out in DMG buffer with 96-well plates at 37?.The reaction was initiated by mixing enzyme with substrates,was ended by mixing Biomol Green Reagen,and was then measured at 620 nm absorbance.5)Co immunoprecipitationHEK-293 cells were co-transfected by plasmids of HA-CDK2(5?g)and Flag-PTPN12(7?g)simultaneously and incubated for 60 hours.The cell lysate were incubated with 25ul HA-beads or Flag-beads with end-to-end rotation overnight at 4?,then beads were washed for 5 times and the immune-precipitated complex protein were subjected to western blotting.6)Assays of cell functionAfter treatment with desired ligands or vehicles,cells were allowed culture for 12 hours or 60 hours.The medium were replaced with 100ul PBS including 500ug/ml MTT and then cultured for another 4 hours.The cultural supernatant was then discarded.The crystals were remelted by addition of 100ul DMSO and 10 minutes vibration and analysed by reading at 490nm absorbance.By MTT assay,we assessed PTPN12 effects on cell proliferation.Cells were digested after starvation for 12 hours in Serum Free Medium.When digestion was terminated,cells were harvested by centrifugation and the supernatant was discarded.Cell pellets were washed by PBS for 1?2 times and re-suspended by serum free medium.Cells were added to upper chamber of 6-well plates(2×106/well)with complete medium containing 25ng/ml EGF at lower chamber.After 36 hours,inserted cells were stained by methanol and crystal violet and counted with the microscope to determine the number of migrated cells.By transwell assay,we assessed PTPN12 effects on cell migration.MDA-MB-231 cells were co-transfected with different PTPN12 catalytic domain constructs and HA-ubiquitin.60 hours after transfection,the cells were stimulated with 100ng/ml EGF for 6 hours under the presence of 10 nM MG 132(a proteasome inhibitor).Then,the cells were lysed on the ice.The soluble lysates were incubated with HA affinity beads for 12 hours at 40?.The HA affinity beads were washed three times by lysis buffer and re-suspended by sample loading buffer.Finally.the immunoprecipitated complexes were subjected for Western Blotting.7)Phosphorylation reaction in vitroThe phosphorylation of PTPN12 by CDK2 was conducted at 37? in MOPS-buffer.There were GST-CDK2,His-PTPN12,as well as ATP and cyclin A in the reaction system.The reaction was started by adding CDK2.The total volume for each reaction was 25?l and the assay were performed in glass tubes.The reaction was finally ended by adding 2×loading buffer and subsequently boiling.Research results1)Crystal structure of the PTPN12 catalytic domain was determined.2)Two structural determinants for substrate specificity of PTPN12 were identified.3)Impact of the Substrate Specificity of PTPN12 on its Cellular Functions was assessed.4)The crystal structure of PTPN12 catalytic domain reveals a CDK2 phosphorylation site.