Identification of Tpr and α-actinin-4 as two novel SLK-interacting proteins

Expression and activity of the Ste20-like kinase, SLR, are increased during kidney development and recovery from ischemia-reperfusion injury. SLK mediates apoptosis in various cells, and can regulate cell cycle progression and cytoskeletal remodeling. In cells, SLK is detected in a high molecular mass complex, suggesting that SLK is a dimer/oligomer, or is in tight association with other proteins. To better understand the regulation, localization and function of SLK, we sought to identify proteins in this high molecular mass complex. Analysis by mass spectroscopy identified the nucleoporin, translocated promoter region (Tpr), and the cytoskeletal protein, alpha-actinin-4, as potential SIX-interacting proteins. Using a protein complementation assay, we showed that the 350 amino acid C-terminal, coiled-coil domain of SLK was responsible for homodimerization, as well as interaction with Tpr and alpha-actinin-4. The association of SLK with Tpr and alpha-actinin-4, respectively, was confirmed by co-immunoprecipitation. Subsets of total cellular SLK colocalized with Tpr at the nuclear envelope, and alpha-actinin-4 in the cytoplasm. Expression of Tpr attenuated activation-specific autophosphorylation of SLK, and blocked SLK-induced apoptosis and AP-1 activity. In contrast to the effect of Tpr, autophosphorylation of SLK was not affected by a-actinin-4. Thus, SLK interacts with Tpr and alpha-actinin-4 in cells, and these protein-protein interactions may control the subcellular localization and the biological activity of SLR. (C) 2015 Elsevier B.V. All rights reserved.