Abstract
Katanin is an ATPase family member protein that participates in microtubule severing. It has heterodimeric structure consisting of 60 kDa (katanin-p60) and 80 kDa (katanin-p80) subunits encoded by KATNA1 and KATNB1 genes, respectively. Katanin-p60 has the enzymatic activity for microtubule severing, whereas katanin-p80 consists of multiple domains with different functions such as targeting katanin-p60 to the centrosome, augmenting microtubule severing by katanin-p60, and even suppressing microtubule severing. Despite the various important functions of katanin-p80, its transcriptional regulation has not been studied yet. Elk1 transcription factor has been shown to interact with microtubules and regulate the transcription of another microtubule severing protein, spastin. In spite of katanin’s importance, and structural and functional similarities to spastin, there is no study on the transcriptional regulation of katanin yet. In this study, we aimed to characterize KATNB1 promoter and analyze the effects of Elk1 on katanin-p80 expression. We identified a 518- bp TATA-less promoter including a critical CpG island and GC boxes as an optimal promoter, and sequential deletion of CpG island and the GC elements gradually decreased the KATNB1 promoter activity. In addition, we showed Elk1 binding on the KATNB1 promoter by EMSA. We found that Elk1 activated KATNB1 promoter, and increased both mRNA and protein levels of katanin-p80 in SH-SY5Y cells. On the other hand, KCl treatment increasing SUMOylation decreased KATNB1 promoter activity. Since microtubule severing is an important cellular mechanism of which malfunctions result in serious diseases such as spastic paraplegia, Alzheimer’s disease and cell cycle related disorders, identification of KATNB1 transcriptional regulation is crucial in understanding the coordination of microtubule severing activity by different proteins in the cells.
Highlights
Microtubule severing is one of the important events in the regulation of microtubule dynamics which is essential for fundamental cellular processes such as cell division and differentiation
In order to define the promoter region that is critical for the expression of KATNB1 gene, promoter deletion fragments were generated at the most feasible intervals according to the presence of the predicted transcription factors binding sites
The deletion constructs of KATNB1 promoter region were designed based on the presence of transcription factor binding sites and GC rich regions, since these sites and regions have important roles in the formation of transcription initiation complex [25]
Summary
Microtubule severing is one of the important events in the regulation of microtubule dynamics which is essential for fundamental cellular processes such as cell division and differentiation. The N-terminal WD40 repeat domain of katanin-p80 is a negative regulator of microtubule severing by katanin-p60 and is necessary for centrosome or spindle pole targeting; while procon domain includes microtubule binding motif and is required for interaction with katanin-p60 and augmentation of its enzymatic activity [3,4,5]. Both katanin-p80 and katanin-p60 are widely distributed throughout the cell, their levels have been found to change independently in different tissue types and developmental stages depending on the demand of microtubule severing. Another study performed in primary hippocampal neurons showed that overexpression of katanin-p60 decreased process numbers, whereas overexpression of kataninp elevated process numbers probably by changing configuration of microtubule array to promote the formation of more processes, presumably by enhancing release of microtubules from the centrosome or increasing the rate of exodus of microtubules from the cell body [6].All these findings indicate that katanin-p80 contributes to the efficacy of katanin-p60 by increasing its severing efficiency, indicating that katanin-p80 has likely other roles in addition to targeting katanin-p60 to the centrosomes [5]
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