Characterization of ankyrin repeat domain 54 (ANKRD54) and its role on the regulation and subcellular localization of Bruton’s tyrosine kinase (BTK)

Bruton's tyrosine kinase (BTK) is an important cytoplasmic signaling protein, where the kinase activity plays a pivotal role in the development, proliferation and differentiation of B-cell lineages. Ankyrin repeat domain 54 (ANKRD54) is a nuclear-resident adaptor protein, where the ankyrin domain repeats are critical for specific protein-protein interaction, while the NLS and NES motifs control the nucleo-cytoplasmic shuttling ability.

We have identified and characterized ANKRD54 as a novel functional (paper I), interaction-partner for BTK using proteomics analysis. ANKRD54 is the first protein identified that specially influences the nuclear export of both BTK and TXK/RLK, in a Crm-1 dependent manner. Further, we mapped the interaction site to the C -terminus of BTK-SH3 domain, by using a synthetic peptide of BTK, covering the following region: C- ARDKNGQEEGYIPSNYVTEAEDS. In addition, tyrosine phosphorylation of BTK was investigated in the presence of increased amount of ANKRD54 and selectively the phosphorylation of BTK was down regulated.

We have presented a second novel interaction-partner and regulator of BTK (paper II), the 14-3-3 ζ protein, which is also identified by proteomics strategy. In this work, we have mapped the interaction sites on BTK to phospho-serine pS51 in the (RGRRGpS)-motif in the PH-domain and phospho-threonine pT495 in the (RHRFQpT)-motif in the kinase domain. Additionally, a newly characterized 14-3-3 inhibitor (BV02) interfered binding with BTK and siRNA knockdown of 14-3-3ζ increased the nuclear translocation of BTK, while overexpression of 14-3-3ζ resulted in accumulation of BTK in the perinuclear region.

We have generated single ankryin domain deletions of ANKRD54 and subsequently characterized their binding capacity and also their influence on the sub-cellular localization of BTK (paper III). In this work, we report that three out of four ankyrin repeats are required for the interaction and nucleo-cytoplasmic shuttling of BTK. Inhibition of Crm-1 nuclear export pathway influences differently the nuclear shuttling; rapid-ANKRD54 versus slow-BTK nuclear accumulation. Furthermore, we have determined that the interaction between BTK and ANKRD54 establishes in the nuclear compartment.

We have classified ANKRD54 as a prime interactor to the SH3-domain of BTK (paper IV). In this study, we utilized a screening strategy based on phage display libraries of the complete human “SH3-domainome” as a possible binding-target for ANKRD54. The aim is to identify the target spectrum and specificity of ANKRD54 for SH3 domain library, containing all the 296 human SH3 domains. The novel finding is that BTK is not only binding to ANKRD54, but also stands out as the preferred interactor, being highly dominant over all other human SH3 domains. However, other lower colony-score candidates for SH3-domain interactions were found, but without any further in vivo/in vitro validation.

List of scientific papers

I. Mohammad DK, Nore BF, Gustafsson MO, Mohamed AJ, Smith CI. Protein Kinase B (AKT) Regulates SYK Activity and Shuttling Through 14-3-3 and Importin 7. Int J Biochem Cell Biol. 2016 Jul 2.
https://doi.org/10.1016/j.biocel.2016.06.024

II. Bestas B, Moreno PM, Blomberg KE, Mohammad DK, Saleh AF, Sutlu T, Nordin JZ, Guterstam P, Gustafsson MO, Kharazi S, Piątosa B, Roberts TC, Behlke MA, Wood MJ, Gait MJ, Lundin KE, El Andaloussi S, Månsson R, Berglöf A, Wengel J, Smith CI., Splice-correcting oligonucleotides restore BTK function in X- linked agammaglobulinemia model. J Clin Invest. 2014 Sep;124(9):4067-81.
https://doi.org/10.1172/JCI76175

Sassi A, Lazaroski S, Wu G, Haslam SM, Fliegauf M, Mellouli F, Patiroglu T, Unal E, Ozdemir MA, Jouhadi Z, Khadir K, Ben-Khemis L, Ben-Ali M, Ben-Mustapha I, Borchani L, Pfeifer D, Jakob T, Khemiri M, Asplund AC, Gustafsson MO, Lundin KE, Falk-Sörqvist E, Moens LN, Gungor HE, Engelhardt KR, Dziadzio M, Stauss H, Fleckenstein B, Meier R, Prayitno K, Maul-Pavicic A, Schaffer S, Rakhmanov M, Henneke P, Kraus H, Eibel H, Kölsch U, Nadifi S, Nilsson M, Bejaoui M, Schäffer AA, Smith CI, Dell A, Barbouche MR1, Grimbacher B. Hypomorphic homozygous mutations in phosphoglucomutase 3 (PGM3) impair immunity and increase serum IgE levels. J Allergy Clin Immunol. 2014 May;133(5):1410-9, 1419.e1-13.
https://doi.org/10.1016/j.jaci.2014.02.025

IV. A. Hussain, Dara K. Mohammed, Manuela O. Gustafsson, Merve Uslu, Abdulrahman Hamasy, Beston F. Nore, Abdalla J. Mohamed and C. I. Edvard Smith. Signaling of the ITK (Interleukin 2-inducible T Cell Kinase)-SYK (Spleen Tyrosine Kinase) Fusion Kinase is Dependent on the Adaptor SLP-76 and on the Adapter Function of the Kinases SYK and ZAP70, J. Biol. Chem., 2013 Mar 8;288(10):7338-50.
https://doi.org/10.1074/jbc.M112.374967