Two such factors that increase the catastrophe rate have recently been identified, namely a kinesin-like protein, XKCM1 Walczak et al. The pMEP4 shuttle vector contains the Epstein-Barr virus origin of replication and the EBNA-1 gene to allow high-copy episomal replication, and the hph gene, which confers hygromycin B resistance in mammalian cells Groger et al.
PCR products were further submitted to Sanger sequencing and subsequently mapped against the genome, confirming correct product amplification. Our genome-scale data analysis and PI3K investigation point to the importance of tubulin autoregulation in normal biology and cancer pathophysiology. Little is known, however, about regulation of MT dynamics in response to external and internal cellular signals. Op18 is also phosphorylated by several kinase systems in response to external signals. Tubulin autoregulation was a central concern in the microtubule field in the s, but it was abandoned as the field moved towards biophysical directions in the s.
During mitosis, Op18 is phosphorylated on all four sites to high stoichiometry; Ser and Ser are phosphorylated by members of the cyclin-dependent kinase family, and Ser and Ser by an as yet unidentified protein kinase Brattsand et al. Cells were incubated overnight. K A proposed model describes the mechanism through which PI3K signaling regulates tubulin gene expression through modification of tubulin autoregulation. DGE and statistical analysis were performed using the edgeR package [ 45 ].
The converse is true for PTX. PI3K inhibition with a small molecule reverses these changes. Our findings, therefore, demonstrate the ubiquity and importance of autoregulation in controlling tubulin gene expression and very likely microtubule biology in general.
Bioinformatic analysis We used the CLIC-gene online tool for bioinformatic analysis of tubulin expression correlation [ 26 ]. Scales of expression profile z-scores are depicted above each heatmap. A cells a kind gift from P. Media with L-glutamine were prepared by adding L-glutamine Corning to the L-glutamine—free medium at 2 mM final concentration.
This effect is achieved through the microtubule-stabilizing activity of PI3K signaling and tubulin autoregulation.
In recent years, pseudogenes have come in the spotlight as potential regulators of their functional counterparts [ 35 ]. For each reference and gene of interest, two sets of primers were designed: one set of primers amplified specifically unspliced pre-mRNA, while the other set of primers amplified specifically spliced mRNA. Using bioinformatics to query public data sets, we provide evidence that coregulation of expressed TUBAs, TUBBs, and TUBGs is observed in response to stimuli other than microtubule-targeting drugs, in tissues, and in cycling and quiescent cell cultures. Prior work on tubulin autoregulation was confined to microtubule-drug—induced effects in a few cell types in culture [ 14 , 36 , 37 ]. Bioinformatic analysis We used the CLIC-gene online tool for bioinformatic analysis of tubulin expression correlation [ 26 ]. Here we show that induced expression of the catalytic subunit of cAMP-dependent protein kinase PKA results in a dramatic increase in cellular MT polymer content concomitant with phosphorylation and partial degradation of Op
Our findings show for the first time, to our knowledge, that tubulin autoregulation via mRNA stability mediates changes in total tubulin concentration triggered by an important signaling pathway. Flow cytometry cell-cycle profiling In the flow cytometry validation experiments, RPE1 hTert cells were grown per treatment condition on cm petri dishes for 1 day for cycling culture and 5 days after reaching full confluency for quiescent cultures. Black and white asterisk represent statistical significance relative to DMSO-treated parental cell line, green asterisks represent statistical significance relative to DMSO-treated control of the same cell line. But if this is the case, why is TUBG1 also coregulated?
Curiously, we reveal that autoregulation controls tubulin pseudogenes despite their lost functionality. Autoregulation suggests functions of tubulin beyond building microtubules A striking feature of our DGE data in quiescent RPE1 hTert cells, which was also seen in two large GEO studies that we reanalyzed, is a lack of coregulation of other microtubule components with tubulin: tubulin autoregulation signature extends to all expressed tubulin but not to microtubule-associated, plus-tip—binding, or motor proteins, nor are these components coregulated on the transcriptional level in response to microtubule damage.
Statistical analysis For gene-expression profiling, statistical analysis was supplied as part of the edgeR package and performed according to the user manual. Each column of the heatmap represents DGE in one condition, labeled on the x-axis. Our genome-scale data analysis and PI3K investigation point to the importance of tubulin autoregulation in normal biology and cancer pathophysiology. These are speculations, but they point out that we truly do not understand the adaptive benefit of tubulin autoregulation despite its frequent occurrence in cellular physiology, as revealed by our bioinformatics analysis. Null module consists of tubulin genes with low Pearson expression correlation.