Receptor-type protein tyrosine phosphatases (RPTPs) are considered to transduce extracellular signals across the membrane through changes in their PTP activity, however, our understanding of the regulatory mechanism is still limited. Here, we show that pleiotrophin (PTN), a natural ligand for protein tyrosine phosphatase receptor type Z (Ptprz) (also called PTPf/ RPTPb), inactivates Ptprz through oligomerization and increases the tyrosine phosphorylation of substrates for Ptprz, G proteincoupled receptor kinase-interactor 1 (Git1) and membrane associated guanylate kinase, WW and PDZ domain containing 1 (Magi1). Oligomerization of Ptprz by an artificial dimerizer or polyclonal antibodies against its extracellular region also leads to inactivation, indicating that Ptprz is active in the monomeric form and inactivated by ligand-induced oligomerization.

Protein-tyrosine phosphatase receptor type Z (Ptprz) is preferentially expressed in the brain as a major chondroitin sulfate proteoglycan. Three splicing variants, two receptor isoforms and one secretory isoform, are known. Here, we show that the extracellular region of the receptor isoforms of Ptprz are cleaved by metalloproteinases, and subsequently the membrane-tethered fragment is cleaved by presenilin/␥-secretase, releasing its intracellular region into the cytoplasm; of note, the intracellular fragment of Ptprz shows nuclear localization. Administration of GM6001, an inhibitor of metalloproteinases, to mice demonstrated the metalloproteinase-mediated cleavage of Ptprz under physiological conditions. Furthermore, we identified the cleavage sites in the extracellular juxtamembrane region of Ptprz by tumor necrosis factor-␣ converting enzyme and matrix metalloproteinase 9. This is the first evidence of the metalloproteinase-mediated processing of a receptor-like protein-tyrosine phosphatase in the central nervous system.Receptor-like protein-tyrosine phosphatases (RPTPs) 2 are a structurally and functionally diverse family of enzymes comprised of eight subfamilies (1). Protein-tyrosine phosphatase receptor type Z (Ptprz, also called PTP or RPTP␤) is a RPTP classified in the R5 subfamily and expressed in neuronal and glial cells in the central nervous system (2-4). The physiological importance of this molecule has been demonstrated through studies of Ptprz-deficient mice (4, 5), which display impairments in hippocampal function in a maturation-dependent manner (6, 7). An independently generated knock-out mouse line suggests a fragility of myelin in the central nervous system (8).It is known that three isoforms of Ptprz are generated by alternative splicing from a single Ptprz gene (on mouse chromosome 6; human chromosome 7), the two transmembrane isoforms Ptprz-A and Ptprz-B and the secretory isoform Ptprz-S (also known as 6B4 proteoglycan or phosphacan) (2, 9 -12), all of which are expressed as chondroitin sulfate proteoglycans in the brain (3). However, some inexplicable issues about the molecular profiles of Ptprz have remained in previous studies. For instance, although there exists substantial expression of the respective transcripts for all isoforms (11, 13), fulllength Ptprz-A has been scarcely observed at the protein level in the adult brain (3, 4). In addition, several lower molecular species have been detected with a specific antibody against Ptprz in wild-type mice (4). The technical difficulty in removal of the chondroitin sulfate chains to separate their core proteins by SDS-PAGE may induce variability in the signal patterns of this molecule in Western blotting among researchers.In this study we examined the molecular profile of Ptprz in the adult mouse brain at both protein and mRNA levels in detail and revealed that the proteolytic fragments are abundantly accumulated. The two receptor isoforms were found to undergo ectodomain cleavage by metalloproteinases, releasing their extracellular fragm...

Inhibition of cyclin-dependent kinases (CDKs) by Thr14 /Tyr 15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G 2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominantnegative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr 14 /Tyr 15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.Cyclins and cyclin-dependent kinases (CDKs) 1 are key regulators of the eukaryotic cell cycle. In mammalian cells, different cyclin-CDK complexes are involved in regulating different cell cycle transitions: cyclin D-CDK4

Nasopharyngeal carcinoma is a rare but highly invasive cancer. As options of agents for effective combination chemoradiotherapy for advanced nasopharyngeal carcinoma are limited, novel therapeutic approaches are desperately needed. The ubiquitin ligase CHFR is known to target PARP1 for degradation and is epigenetically inactivated in nasopharyngeal carcinoma. We present evidence that PARP1 protein is indeed overexpressed in nasopharyngeal carcinoma cells in comparison with immortalized normal nasopharyngeal epithelial cells. Tissue microarray analysis also indicated that PARP1 protein is significantly elevated in primary nasopharyngeal carcinoma tissues, with strong correlation with all stages of nasopharyngeal carcinoma development. We found that the PARP inhibitor AZD2281 (olaparib) increased DNA damage, cell-cycle arrest, and apoptosis in nasopharyngeal carcinoma cells challenged with ionizing radiation or temozolomide. Isobologram analysis confirmed that the cytotoxicity triggered by AZD2281 and DNAdamaging agents was synergistic. Finally, AZD2281 also enhanced the tumor-inhibitory effects of ionizing radiation in animal xenograft models. These observations implicate that PARP1 overexpression is an early event in nasopharyngeal carcinoma development and provide a molecular basis of using PARP inhibitors to potentiate treatment of nasopharyngeal carcinoma with radio-and chemotherapy.

The current paradigm states that exit from mitosis is triggered by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) acting in concert with an activator called CDC20. While this has been well established for a number of systems, the evidence of a critical role of CDC20 in somatic cells is not unequivocal. In this study, we reexamined whether mitotic exit can occur properly after CDC20 is depleted. Using single-cell analysis, we found that CDC20 depletion with small interfering RNAs (siRNAs) significantly impaired the degradation of APC/C substrates and delayed mitotic exit in various cancer cell lines. The recruitment of cyclin B1 to the core APC/C was defective after CDC20 downregulation. Nevertheless, CDC20-depleted cells were still able to complete mitosis, albeit requiring twice the normal time. Intriguingly, a high level of cyclin-dependent kinase 1 (CDK1)-inhibitory phosphorylation was induced during mitotic exit in CDC20-depleted cells. The expression of an siRNA-resistant CDC20 rescued both the mitotic exit delay and the CDK1-inhibitory phosphorylation. Moreover, the expression of a nonphosphorylatable CDK1 mutant or the downregulation of WEE1 and MYT1 abolished mitotic exit in CDC20-depleted cells. These findings indicate that, in the absence of sufficient APC/C activity, an alternative mechanism that utilized the classic inhibitory phosphorylation of CDK1 could mediate mitotic exit.Cyclin-dependent kinase 1 (CDK1) (also called CDC2) is one of the key protein kinases for promoting mitosis. The activation of CDK1 requires binding to its activating partner (cyclin B1) and the phosphorylation of a residue on the T-loop (Thr161). While CDK1 Thr161 phosphorylation occurs after cyclin B1 binding, cyclin B1 itself oscillates during the cell cycle, accumulating from S phase, and is destroyed at the end of mitosis (reviewed in reference 10).Before mitosis, cyclin B1-CDK1 complexes are kept in a CDK1 Thr14/Tyr15 -phosphorylated and inactive state by two kinases called WEE1 and MYT1. While WEE1 specifically phosphorylates CDK1 Tyr15 (29), the endoplasmic reticulum-/Golgi complex-located MYT1 displays a stronger preference for CDK1 Thr14 (4,19). WEE1 itself is regulated by several kinases. WEE1Ser123 is phosphorylated by CDK1 at the onset of mitosis, thereby generating a binding motif to allow PLK1 to phosphorylate WEE1 Ser53 (45,47). The phosphorylation of WEE1 Ser123 also independently primes the phosphorylation of WEE1 Ser121 by CK2 (46). Together, phosphorylated Ser123, Ser121, and Ser53 serve as phosphodegrons that target WEE1 for degradation by the ubiquitin ligase SCF ␤-TrCP (46), thereby ensuring that WEE1 activity is suppressed during mitosis. Similarly, MYT1 activity decreases during mitosis, coinciding with the phosphorylation by PLK1 and CDK1 (4,26,50). At the end of G 2 phase, the stockpile of inactive cyclin B1-CDK1 complexes is activated by members of the CDC25 family.With the feedback loops that simultaneously activate CDC25 and inactivate WEE1/MYT1 (reviewed in reference 18), the ac...

Inhibition of cyclin-dependent kinase 1 (CDK1) by phosphorylation is a key regulatory mechanism for both the unperturbed cell cycle and the DNA damage checkpoint. Although both WEE1 and MYT1 can phosphorylate CDK1, little is known about the contribution of MYT1. We found that in contrast to WEE1, MYT1 was not important for the normal cell cycle or checkpoint activation. Time-lapse microscopy indicated that MYT1 did, however, have a rate-determining role during checkpoint recovery. Depletion of MYT1 induced precocious mitotic entry when the checkpoint was abrogated with inhibitors of either CHK1 or WEE1, indicating that MYT1 contributes to checkpoint recovery independently of WEE1. The acceleration of checkpoint recovery in MYT1-depleted cells was due to a lowering of threshold for CDK1 activation. The kinase activity of MYT1 was high during checkpoint activation and reduced during checkpoint recovery. Importantly, although depletion of MYT1 alone did not affect long-term cell growth, it potentiated with DNA damage to inhibit cell growth in clonogenic survival and tumor xenograft models. These results reveal the functions of MYT1 in checkpoint recovery and highlight the potential of MYT1 as a target for anti-cancer therapies.

Protein-tyrosine phosphatase receptor type Z (Ptprz) has multiple substrate proteins, including G protein-coupled receptor kinase-interactor 1 (Git1), membrane-associated guanylate kinase, WW and PDZ domain-containing 1 (Magi1), and GTPase-activating protein for Rho GTPase (p190RhoGAP). We have identified a dephosphorylation site at Tyr-1105 of p190RhoGAP; however, the structural determinants employed for substrate recognition of Ptprz have not been fully defined. In the present study, we revealed that Ptprz selectively dephosphorylates Git1 at Tyr-554, and Magi1 at Tyr-373 and Tyr-858 by in vitro and cell-based assays. Of note, the dephosphorylation of the Magi1 Tyr-858 site required PDZ domain-mediated interaction between Magi1 and Ptprz in the cellular context. Alignment of the primary sequences surrounding the target phosphotyrosine residue in these three substrates showed considerable similarity, suggesting a consensus motif for recognition by Ptprz. We then estimated the contribution of surrounding individual amino acid side chains to the catalytic efficiency by using fluorescent peptides based on the Git1 Tyr-554 sequence in vitro. The typical substrate motif for the catalytic domain of Ptprz was deduced to be Glu/Asp-Glu/Asp-Glu/Asp-Xaa-Ile/ Val-Tyr(P)-Xaa (Xaa is not an acidic residue). Intriguingly, a G854D substitution of the Magi1 Tyr-858 site matching better to the motif sequence turned this site to be susceptible to dephosphorylation by Ptprz independent of the PDZ domainmediated interaction in cells. Furthermore, we found by database screening that the substrate motif is present in several proteins, including paxillin at Tyr-118, its major phosphorylation site. Expectedly, we verified that Ptprz efficiently dephosphorylates paxillin at this site in cells. Our study thus provides key insights into the molecular basis for the substrate recognition of Ptprz.Protein-tyrosine phosphorylation is a dynamic process governed by the balanced actions of protein-tyrosine kinases (PTKs), 2 and protein-tyrosine phosphatases (PTPs), and critical to the regulation of numerous physiological processes (for review, see Ref. 1). The specificity of the signal transduction depends on the ability of each PTK or PTP to phosphorylate or dephosphorylate precisely particular sites on specific substrates, respectively. Elucidation of the specificity for individual PTPs has been an important subject of investigation; however, even the identification of PTP substrates is still methodologically difficult. To our knowledge, substrate specificity of PTPs has been characterized only for PTP1B (2).Receptor-like PTPs (RPTPs) are a structurally and functionally diverse family of enzymes comprised of eight subfamilies. PTP receptor type Z (Ptprz, also called PTP or RPTP␤) is a RPTP classified in the R5 subfamily together with Ptprg (PTP␥). Three isoforms of Ptprz are generated by alternative splicing from a single Ptprz gene: the two transmembrane isoforms Ptprz-A and Ptprz-B and the secretory isoform Ptprz-S (also known as phosphacan...

Cell cycle checkpoints that monitor DNA damage and spindle assembly are essential for the maintenance of genetic integrity, and drugs that target these checkpoints are important chemotherapeutic agents. We have examined how cells respond to DNA damage while the spindle-assembly checkpoint is activated. Single cell electrophoresis and phosphorylation of histone H2AX indicated that several chemotherapeutic agents could induce DNA damage during mitotic block. DNA damage during mitotic block triggered CDC2 inactivation, histone H3 dephosphorylation, and chromosome decondensation. Cells did not progress into G1 but seemed to retract to a G2-like state containing 4N DNA content, with stabilized cyclin A and cyclin B1 binding to Thr14/Tyr15-phosphorylated CDC2. The loss of mitotic cells was not due to cell death because there was no discernible effect on caspase-3 activation, DNA fragmentation, or viability. Extensive DNA damage during mitotic block inactivated cyclin B1-CDC2 and prevented G1 entry when the block was removed. The mitotic DNA damage responses were independent of p53 and pRb, but they were dependent on ATM. CDC25A that accumulated during mitosis was rapidly destroyed after DNA damage in an ATM-dependent manner. Ectopic expression of CDC25A or nonphosphorylatable CDC2 effectively inhibited the dephosphorylation of histone H3 after DNA damage. Hence, although spindle disruption and DNA damage provide conflicting signals to regulate CDC2, the negative regulation by the DNA damage checkpoint could overcome the positive regulation by the spindle-assembly checkpoint.