Tumor-Derived Extracellular Mutations of PTPRT/PTPR Are Defective in Cell Adhesion
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Tumor-Derived Extracellular Mutations of PTPRT/PTPR Are Defective in Cell Adhesion Jianshi Yu,1,2 Scott Becka,3 Peng Zhang,2 Xiaodong Zhang,1,2 Susann M. Brady-Kalnay,1,3,4 and Zhenghe Wang1,2,5 1Case Comprehensive Cancer Center and Departments of 2Genetics, 3Molecular Biology and Microbiology, and 4Neurosciences, School of Medicine, Case Western Reserve University; and 5Genomic Medicine Institute, Cleveland Clinic Foundation, Cleveland, Ohio Abstract nonsense mutations and frameshifts, suggested that these Receptor protein tyrosine phosphatase T (PTPRT/PTPR) mutations were inactivating (2). Biochemical analyses showed is frequently mutated in human cancers including colon, that missense mutations in the catalytic domains of PTPU lung, gastric, and skin cancers. More than half of the diminished its phosphatase activity, and overexpression of identified tumor-derived mutations are located in the PTPU inhibited colorectal cancer cell growth (2). Taken extracellular part of PTPR. However, the functional together, these studies strongly supported the notion that PTPU significance of those extracellular domain mutations normally acts as a tumor suppressor gene. This conclusion was remains to be defined. Here we report that the also supported by a transposon-based somatic mutagenesis extracellular domain of PTPR mediates homophilic screen in mice, in which PTPU was isolated as a target gene cell-cell aggregation. This homophilic interaction is very from two different mouse transgenic sarcomas (3). specific because PTPR does not interact with its closest PTPRT (PTPU) is a member of the type IIB receptor protein homologue, PTPM, in a cell aggregation assay. We tyrosine phosphatase (RPTP) subfamily (4). Other members of further showed that all five tumor-derived mutations this subfamily include PTPRM (PTPA), PTPRK (PTPn), and E k located in the NH2-terminal MAM and immunoglobulin PCP2 (also called PTP , PTPc, PTPRO-omicron, PTP ,or domains impair, to varying extents, their ability to form hPTP-J; ref. 5). These four RPTPs share the same domain cell aggregates, indicating that those mutations are structure: an extracelluar domain, a juxtamembrane region, and loss-of-function mutations. Our results suggest that two phosphatase domains (6). The extracellular domains of type PTPR may play an important role in cell-cell adhesion IIB RPTPs have high sequence identities (6-8), all consisting and that mutational inactivation of this phosphatase of a MAM (memprin/A5/PTPA) domain, an immunoglobulin could promote tumor migration and metastasis. domain, and four fibronectin type III repeats (6). The MAM (Mol Cancer Res 2008;6(7):1106–13) domain is suggested to play a role in protein dimerization (6). The immunoglobulin domain is a disulfide structure that is found in many cell surface proteins and has been shown to Introduction mediate homophilic and heterophilic interactions between cell Tyrosine phosphorylation is coordinately controlled by adhesion molecules (6). The fibronectin type III motif was protein tyrosine kinases and phosphatases and is a central originally identified in the extracellular matrix protein fibro- feature of many signaling pathways involved in tumor nectin and later found to be present in many immunoglobulin development (1). Whereas activating mutations in protein superfamily cell adhesion molecules (6). We have identified tyrosine kinases have been shown to play vital roles in f15 somatic mutations that are localized in the extracellular tumorigenesis (1), the role of phosphatases is less well defined. domain of PTPRT/PTPU. How these mutations affect the U We recently identified PTPRT, also known as PTP , as the most functions of PTPU remains to be determined. frequently mutated PTP gene in colorectal cancers (2). PTPRT Three close homologues of PTPU [PTPA, PTPn, and PTPE was also mutationally altered in lung cancer, gastric cancer, (PCP-2)] are known to mediate homophilic cell-cell adhesion and melanomas (2). The spectrum of mutations, which included (9-12). Expression of the full-length PTPA (9, 10) or a construct encoding the extracellular, transmembrane, and 55 amino acids of the juxtamembrane domain of PTPA in nonadhesive Sf9 insect cells induces cell aggregation (9). Furthermore, homo- Received 10/29/07; revised 2/29/08; accepted 4/9/08. A A Grant support: NIH grants R01-CA127590 and U54CA116867 (Z. Wang) and philic binding of PTP was shown between PTP -coated grants R01-EY12251 and RO1-NS051520 (S.M. Brady-Kalnay), the Concern fluorescent beads and cells that endogenously express PTPA Foundation, the V Foundation (Z. Wang), and Visual Sciences Research Center (9). The minimal region required for homophilic binding was Core Grant PO-EY11373 from the National Eye Institute (S.M. Brady-Kalnay). A The costs of publication of this article were defrayed in part by the payment of mapped to the immunoglobulin domain of PTP (13). In page charges. This article must therefore be hereby marked advertisement in addition, the immunoglobulin domain is required for proper cell accordance with 18 U.S.C. Section 1734 solely to indicate this fact. surface localization (14). However, assays that test cell-cell Requests for reprints: Zhenghe Wang, Department of Genetics and Case Comprehensive Cancer Center, Case Western Reserve University, Wolstein aggregation show that the MAM and immunoglobulin domains Research Building 3-120, 10900 Euclid Avenue, Cleveland, OH 44106. Phone: as well as the first two fibronectin type III repeats are required 216-368-0446; Fax: 216-368-8919. E-mail: [email protected] n Copyright D 2008 American Association for Cancer Research. for efficient cell-cell aggregation (15-18). Similarly, PTP was doi:10.1158/1541-7786.MCR-07-2123 also shown to mediate homophilic cell-cell aggregation (11). 1106 Mol Cancer Res 2008;6(7). July 2008 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2008 American Association for Cancer Research. PTPRT Mutations Are Defective in Cell Adhesion 1107 Here we report that PTPRT (PTPU), like its homologues, sion of single cells (Fig. 2). These results indicate that the mediates homophilic cell-cell aggregation in Sf9 cells. Most extracellular fragment of PTPU plays an important role in importantly, the tumor-derived mutations located in the MAM mediating cell-cell aggregation and that the phosphatase and immunoglobulin domains cause defective cell-cell adhesion activity of PTPU is not required for this function. Surprisingly, function. although the construct that encodes the PTPU extracellular fragment with the transmembrane domain sequences only Results (Extra-TM) was expressed on the cell surface (see Fig. 7), this The Extracellular Domain of PTPq Mediates Cell-Cell protein failed to mediate cell aggregation of Sf9 cells (Fig. 2), Aggregation in Sf9 Cells suggesting that additional juxtamembrane sequences are To test whether PTPU mediates homophilic cell-cell required for either forming or stabilizing the cell aggregates. adhesion, we made PTPU baculoviral constructs expressing (a) full-length PTPU;(b) the intracellular fragment of PTPU PTPq Mediates Highly Specific Homophilic Cell-Cell (Intra); (c) the extracellular fragment of PTPU with its Aggregation transmembrane domain (Extra-TM); (d) the extracellular To test whether PTPU mediates homophilic cell-cell fragment of PTPU with its transmembrane, juxtamembrane, aggregation, one set of Sf9 cells was infected with viruses and wedge domains (Extra-JMD-W); and (e)achimera expressing enhanced green fluorescent protein and another set containing the extracellular and transmembrane domains of of Sf9 cells was coinfected with viruses expressing the epidermal growth factor receptor and the intracellular PTP the full-length PTPU and red fluorescent protein. The green domains of PTPU (Fig. 1A). All of those fragments were tagged fluorescent protein–expressing cells were mixed with equal with V5 epitope tags. Proteins of the expected size were number of cells expressing full-length PTPU (coexpressing red expressed in Sf9 cells infected with those baculoviral constructs fluorescent protein) and the aggregation assays were done. (Fig. 1B). All of these proteins, but the intracellular PTPU, were As shown in Fig. 3A, the green fluorescent protein–labeled expressed on the surface on Sf9 cells (Figs. 1C and 7). To test cells did not aggregate and did not contribute to the aggregates whether these PTPU recombinant proteins mediate cell-cell formed by cells expressing full-length PTPU (red fluorescent adhesion, a cell aggregation assay was done following the protein). These results strongly suggest that the aggregation of procedure of Brady-Kalnay et al. (9). Sf9 cells infected with a Sf9 cells expressing PTPU is mediated through a homophilic baculovirus encoding PTPA, which was previously shown to interaction among PTPU molecules in trans, but not through a mediate cell-cell aggregation, were treated in parallel as a heterophilic interaction between molecules expressed on the positive control (Fig. 2). As shown in Fig. 2, the nonadhesive surface of Sf9 cells. Interestingly, the homophilic interaction Sf9 cells expressing either the full-length PTPU or PTPU-JMD- of PTPU is very specific. Although 63% of the amino acid W formed cell aggregates. On the contrary, Sf9 cells expressing sequences of the extracellular domain of PTPU are identical either the intracellular fragments of PTPU or the epidermal with that of PTPRM (PTPA), Sf9 cells expressing PTPU growth factor receptor/PTPU chimeras remained as a suspen- (coexpressing red fluorescent