Binding partners of the kinase domains inDrosophilaobscurin and their effect on the structure of the flight muscle

Abstract

Drosophila obscurin (Unc-89) is a titin-like protein in the M-line of the muscle sarcomere. Obscurin has two kinase domains near the C-terminus, both of which are predicted to be inactive. We have identified proteins binding to the kinase domains. Kinase domain 1 bound Bällchen (Ball, an active kinase), and both kinase domains 1 and 2 bound MASK (a 400-kDa protein with ankyrin repeats). Ball was present in the Z-disc and M-line of the indirect flight muscle (IFM) and was diffusely distributed in the sarcomere. MASK was present in both the M-line and the Z-disc. Reducing expression of Ball or MASK by siRNA resulted in abnormalities in the IFM, including missing M-lines and multiple Z-discs. Obscurin was still present, suggesting that the kinase domains act as a scaffold binding Ball and MASK. Unlike obscurin in vertebrate skeletal muscle, Drosophila obscurin is necessary for the correct assembly of the IFM sarcomere. We show that Ball and MASK act downstream of obscurin, and both are needed for development of a well defined M-line and Z-disc. The proteins have not previously been identified in Drosophila muscle.

Obscure functions: the location–function relationship of obscurins

“…In the mature fly, four isoforms (A, B, C, and D) are expressed in the thorax, while two of these isoforms (A and C) are also expressed in the indirect flight muscles (IFM). On the contrary, the larva expresses only obscurin isoform-E (Katzemich et al 2012(Katzemich et al , 2015. Unfortunately, their protein composition is not well annotated and lacks experimental confirmation; herein we provide the domain architecture for each predicted isoform (Fig 1).…”

“…More recently, the Drosophila homolog of obscurin has also been identified ( Fig. 1) (Katzemich et al 2012(Katzemich et al , 2015. Specifically, five obscurin isoforms are expressed in Drosophila in a developmental and tissue-specific manner.…”

“…Drosophila obscurin isoforms possess a common COOHterminal domain, which includes two FnIII domains an Ig domain and two pseudo-kinase domains. Unlike mammalian obscurin-A, there is no analogous ankyrin-binding domain in Drosophila obscurin or C. elegans UNC-89 (Katzemich et al 2012(Katzemich et al , 2015.…”

“…The dense bodies are anchored in the muscle membrane and function to link the actin filaments to the extracellular matrix through connections at Z-discs and costameres. In Drosophila, obscurin concentrates within the sarcomere at the M-bands (Katzemich et al 2015). The multiple subcellular distributions of this family of proteins gives rise to multiple functions for obscurins.…”

“…Within the sarcomere at the level of the M-band, mammalian obscurins interact with titin, myomesin, ankyrin-B, Ras homolog gene family, member A (RhoA), and slow skeletal myosin binding protein C variant 1 (sMyBP-Cv1) (Perry et al 2013). In C. elegans at the M-band, UNC-89 interacts with copine domain protein atypical-1 (CPNA-1), UNC-15 (paramyosin), small CTD-phosphatase-like-1 (SCPL-1), four and a half LIM domains protein 9 (LIM9/FHL), maternal effect lethal 26 (MEL-26), and Rho1 (Gieseler et al 2016;Qadota et al 2008a, b;Warner et al 2013;Wilson et al 2012) and Drosophila obscurins interact with Ball and multiple ankyrin repeats single KH domain (MASK) at the M-band (Katzemich et al 2015). In addition, mammalian obscurins interact with the NH 2 -terminus of titin and Ran-binding protein 9 (RanBP9) at Z-discs (Bowman et al 2008), small ankyrin-1 at the SR , and ankyrin-B at costameres (Randazzo et al 2013).…”