The Journal of Neuroscience, November 16, 2005, ():
PBK/TOPK, a Proliferating Neural Progenitor-Specific Mitogen-Activated Protein Kinase Kinase
J. Neurosci. Dougherty et al.
25: 10773
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1: PBK/TOPK protein structure and expression in tumors suggests role in late cell-cycle.
A) PBK/TOPK was detectably expressed in 79 of 85 tumors by microarray analysis. To form hypothesis about the function of PBK/TOPK, we looked at the function of its correlates in this data set. Bars on graph represent functional classification by GO biological process for top 100 PBK/TOPK-correlated (blue, with correlations ranging from .93 to .42) and –anti-correlated (yellow) genes. 46 of 100 correlated genes and 21 of 100 anti-correlated genes were ‘known genes’ that could be categorized. Of these 46 genes, 24 were involved in the cell-cycle (blue arrow). EASE was used to test for statistical overrepresentation of categories. Categories related to the cell-cycle were the most significant for correlates, and processes related to ion transport were most significant for anti-correlates. * = p<.05, ** = p<.005, ***=p<10e-10. B) Schematic of PBK/TOPK protein. Position of kinase domains is shown in gray, cyclinB/CDK1 phosphorylation site is shown in blue, aspartic acid rich region is shown in orange, and C terminal PDZ-binding motif in yellow. C) Multiple species alignment reveals conservation of cyclinB/CDK1 phosphorylation site, suggesting importance of this cell-cycle motif. Sections of alignments for eleven species of vertebrates. Color-highlighted regions correspond to B. Boxes show matches to consensus. cyclinB/CDK1 site (T/S-P-X-K/R) is conserved in 10 of 11 species, but C-terminal PDZ-binding domain is not.
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Supplemental Figure 2: Neural progenitors also express PBK/TOPK and require P38 activity for normal proliferation.
A) A subset of cultured multipotent neural progenitors cultured from E12 telencephalon are Lex positive (green) and also express PBK/TOPK (red), which is most clearly detectable during mitosis. B) In these cells, there is a SB203580 dose dependant decrease in proliferation as assessed by cell number. C) Forty eight hours of exposure to highest dose of drug resulted in DNA aneuploidy. Dot plots: cell-cycle assessment in progenitors as measured by measure of area (FL2-A) and width (FL2-W) of propridium iodide staining of DNA content. Yellow circle indicates normally cycling cells. Blue circle indicates cells with DNA aneuploidy, which have slightly more DNA (total area) than normal G1 cells but with unusual signal width. Histogram derived from dot plots shows a normal cell-cycle profile and one treated with drug. DNA aneuploidy here is seen as the shoulder indicated by blue arrow. Note also increase in debris and decrease in S phase cells.
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Supplemental Figure 3: PBK/TOPK expressed in EGF-R and NG2 positive cells in adult mouse brain.
A) Schematics of tissue slices showing locations of B and C in red boxes. B) Almost all EGFR positive cells are PBK/TOPK positive in the SEZ. C) Some PBK/TOPK positive cells in the SEZ are NG2 positive (yellow arrows), though there are many NG2 positive cells outside of the SEZ (bottom right panel), and some in the SEZ (blue arrow) that are clearly PBK/TOPK negative.
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Supplemental Figure 4: Model of PBK/TOPK expression in adult neurogenesis.
Past evidence and our current studies suggest that there is a quiescent population of GFAP positive, PBK/TOPK negative stem cells (blue) in the adult subependymal zone that can be recruited to the cell-cycle. These cells give rise to PBK/TOPK positive, GFAP negative, rapidly proliferating cells (red), that in turn, gives rise to PBK/TOPK negative, DCX positive post-mitotic immature neurons (green). Relative amount of amplification of rapidly proliferating progenitor cell is unknown, and it is also unknown if progeny of PBK/TOPK positive cells can become glia, or revert to quiescent stem cells. This diagram only includes markers investigated here.
