ASB9 interacts with ubiquitous mitochondrial creatine kinase and inhibits mitochondrial function

Abstract

Background: The ankyrin repeat and suppressor of cytokine signalling (SOCS) box proteins (Asbs) are a large protein family implicated in diverse biological processes including regulation of proliferation and differentiation. The SOCS box of Asb proteins is important in a ubiquitination-mediated proteolysis pathway. Here, we aimed to evaluate expression and function of human Asb-9 (ASB9). Results: We found that a variant of ASB9 that lacks the SOCS box (ASB9ΔSOCS) was naturally detected in human cell lines but not in peripheral blood mononuclear cells or normal hepatocytes. We also identified ubiquitous mitochondrial creatine kinase (uMtCK) as a new target of ASB9 in human embryonic kidney 293 (HEK293) cells. The ankyrin repeat domains of ASB9 can associate with the substrate binding site of uMtCK in a SOCS boxindependent manner. The overexpression of ASB9, but not ASB9ΔSOCS, induces ubiquitination of uMtCK. ASB9 and ASB9ΔSOCS can interact and colocalise with uMtCK in the mitochondria. However, only expression of ASB9 induced abnormal mitochondrial structure and a decrease of mitochondrial membrane potential. Furthermore, the creatine kinase activities and cell growth were significantly reduced by ASB9 but not by ASB9ΔSOCS. Conclusions: ASB9 interacts with the creatine kinase system and negatively regulates cell growth. The differential expression and function of ASB9 and ASB9ΔSOCS may be a key factor in the growth of human cell lines and primary cells.

Protein Interaction Screening for the Ankyrin Repeats and Suppressor of Cytokine Signaling (SOCS) Box (ASB) Family Identify Asb11 as a Novel Endoplasmic Reticulum Resident Ubiquitin Ligase

“…Furthermore, the presence of proteins like ubiquitin, NEDD8, and CAND1 underscores the notion that the expressed tagged forms of ASBs form active and dynamic E3 complexes (24). Importantly, we were able to confirm the association of ASB9 to the creatine Kinase b as well as the association of ASB9 to the ubiquitous mitochondrial creatine kinase, as reported previously (12,13).…”

“…For example, ASB2 is up-regulated by retinoic acid in acute promyelocytic cells where it targets filament A and B for proteasomal degradation, thereby regulating hematopoetic cell differentiation (10). ASB3 negatively regulates the TNF-R2 pathway leading to the ablation of JNK activation (11), whereas Asb9 is a regulator of creatine kinase B as well as the mitochondrial form of creatine kinase (12,13). Furthermore, the zebra fish counterpart of the mammalian ASB11 (d-ASB11) is a positive regulator of Notch signaling in vivo and influences the neural progenitor compartment of the embryos.…”

The SOCS box—Adapting proteins for ubiquitination and proteasomal degradation

“…Asb9 targets creatine kinase B (CKB) and a mitochondrial form of CK (uMtCK) for degradation in a SOCS box-dependent manner. Interestingly, a natural Asb9 splice variant lacking the SOCS box was recently identified in human cell lines and may function in a dominant negative manner to promote cell growth (65,66).…”

Expression and characterization of transmembrane and coiled-coil domain family 3

“…The resulting peptides were analyzed using electrospray ionization-time of flight mass spectrometry/mass spectrometry (ESI-TOF MS/MS). This was done using a Micromass Q-TOF MA equipped with a nano-spray source (Waters, Milford, MA, USA) in an In2gen facility (Seoul, Korea), as previously described (15). The amino acid sequences of the resulting peptides were analyzed using the database from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov).…”

“…After removal of the cell debris by centrifugation, the cell lysates were mixed with the anti-Myc-tag antibody for 2 h at 4 o C. Immuno-complexes containing TMCC3 and anti-Myc-tag antibody was collected by Protein A-Sepharose CL-4B (10% (v/v) slurry, Amersham Pharmacia Biotech) as previously described (15).…”