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Malignant melanoma continues to pose a substantial clinical challenge and better understanding of melanoma on the molecular level is critical for improved management of this disease. Of relevance to cancer and melanoma in particular, liprins contribute to the process of invasion and metastasis, possibly through their interaction with the metastasis-associated protein S100A4[3,4], which is expressed in melanoma. Liprins are a family of leukocyte common antigen-related (LAR) transmembrane protein tyrosine phosphatase interacting proteins. Based on sequence similarity and binding characteristics, liprins are subdivided into α-type liprins and β-type liprins. Liprin α-1 was originally identified by virtue of its ability to bind to the cytoplasmic phosphatase domain of the transmembrane tyrosine phosphatase LAR. Liprin β-1 and -2 were identified as α-liprin binding proteins. Here we present novel findings documenting increased abundance of liprin β-1 in melanoma and its strong association with Kank1 and Kank2 proteins which are involved in suppression of cellular proliferation and regulation of cell migration[6-8]. Interaction with Kank proteins implicates liprin β-1 in molecular events driving the tumor biology of melanoma.
Protein abundance of liprin β-1 is increased in melanoma while its role remains unknown. We employed the strategy of interacting partner identification to investigate the role of liprin β-1 in melanoma. Possible functions of liprin β-1 may be hypothesized when considering the known functions of its interacting proteins.
Interacting partners of liprin β-1 in the melanoma cell line SK-MEL-28 were co-immunoprecipitated and subjected to mass spectrometry analysis. Select interacting partners of liprin β-1 were confirmed by Western blotting. Finally, binding regions of liprin β-1 and two interacting partners Kank1 and Kank2 were identified by testing the binding characteristics of truncations.
To test whether liprin β-1 is potentially involved in the tumor biology of melanoma, we evaluated liprin β-1 abundance by Western Blot in a panel of established melanoma cell lines. Figure 1A shows significantly elevated liprin β-1 abundance in melanoma cell lines compared to NHM.
The role of liprin β-1 in melanoma is unknown. To address this, we investigated potential molecular interaction partners of liprin β-1 in SK-MEL-28 cells. Endogenous liprin β-1 was immunoprecipitated and subjected to SDS-PAGE. Protein bands were excised at 10 slices per gel lane and digested by trypsin in situ. The digested peptides were subjected to HPLC-ESI-MS/MS analysis. Mass spectrometry identified a number of proteins which co-immunoprecipitated with liprin β-1 including Kank1 and Kank2 (Table S).
Kank proteins have recently been implicated in suppression of cellular proliferation and regulation of actin cytoskeleton. As such they represent interesting and plausibly relevant binding partners of liprin β-1. We therefore investigated further the observed liprin β-1 interaction with Kanks. First we confirmed the interaction by Western blotting. Endogenous liprin β-1, Kank1 or Kank2 were immunoprecipitated from SK-MEL-28 cells, and immunoblotted with either anti-liprin β-1, anti-Kank1 or anti-Kank2 antibody. The results clearly demonstrated that liprin β-1 strongly associates with Kank1 and Kank2 in a complex that can be immunoprecipitated with either liprin β-1 or Kank antibodies (Figure 1B and 1C). SK-MEL-2 and WM-266-4 cell lines produce the same results (Figure S1)
The study of protein-protein interaction can be facilitated by ectopic expression of tagged proteins of interest in cultured cells. To employ this approach, we first evaluated six melanoma cell lines for the ability to express an HA tagged liprin β-1 construct. SK-MEL-2 was chosen for further studies of liprin β-1 - Kank interactions (Figure S2). To identify liprin β-1 domains involved in the interaction with Kank1 and Kank2, we generated DDK-tagged liprin β-1 expression constructs including full-length liprin β-1 (Lipβ-F), along with three truncated liprin β-1 constructs expressing the N-terminus: (Lipβ-N), which contains the α-helical coiled-coil segment functional domain, the central region (Lipβ-M) and the C-terminus (Lipβ-C) (see Data S1). SK-MEL-2 cells were transfected with vector (control), liprin β-1 full length, or one of the three truncations. The cells were harvested, and the lysates were immunoprecipitated with anti-DDK antibody. Immunoprecipitates were subjected to Western Blotting and probed with anti-Kank1, anti-Kank2, or anti-DDK antibody. Figure 2A shows that both Kank1 and Kank2 bind to full-length liprin β-1 and Lipβ-N, but not to Lipβ-M or Lipβ-C, indicating that the association is mediated by the N-terminus of liprin β-1. WM-266-4 cells produce the same results (Figure S3)
To determine which region of Kank1 binds to liprin β-1, we followed the approach described above. We generated myc-tagged constructs expressing either full length Kank1 (Kank1-F), the N-terminal region of Kank1 (Kank1-N), which contains the α-helical coiled-coil segment functional domain, the central region of Kank1 (Kank1-C) and the C-terminal region of Kank1 (Kank1-C). Similarly, we generated HA-tagged Kank2 constructs, including full length Kank2 (Kank2-F), the N-terminal region of Kank2 (Kank2-N), the central region of Kank2 (Kank2-C) and the MC region of Kank2 (Kank2-MC). Expression of these constructs in SK-MEL-2 cells followed by co-immunoprecipitation demonstrated that only full length and N-terminal constructs of Kank1 and Kank2 bind endogenous liprin β-1 (Figure 2B and 2C). WM-266-4 cells produce the same results (Figure S4). This suggests that it is the N-terminal, coil-coil domain of Kank proteins that mediates their interaction with liprin β-1.
We have demonstrated significantly increased liprin β-1 protein abundance in melanoma cells as compared to normal human melanocytes (NHM). Further, we have identified a number of potential interacting partners of liprin β-1, including Kank1 and Kank2. The association of liprin β-1 and Kank proteins is mediated through the N-terminal domain of both liprin β-1 and Kank proteins. For further discussion, see Data S2.
ML and LJM designed the study; AEM and ML performed the experiments; ML, AEM and AS analyzed the data and wrote the manuscript. This work was supported by NIH Grants R01DK47936.
Disclosure Statement: The authors have nothing to declare.