Abstract
BackgroundTartrate–resistant acid phosphatase (TRAP/ ACP5) belongs to the binuclear metallophosphatase family and is present in two isoforms. The primary translation product is an uncleaved TRAP 5a isoform with low phosphatase activity. TRAP 5a can be post-translationally processed to a cleaved TRAP 5b isoform with high phosphatase activity by e.g. cysteine proteinases, such as Cathepsin K (CtsK). The relevance of the phosphatase activity of TRAP 5b has been demonstrated for proliferation, migration and invasion of cancer cells. TRAP-overexpressing MDA-MB-231 breast cancer cells displayed higher levels of TRAP 5a and efficient processing of TRAP 5a to TRAP 5b protein, but no changes in levels of CtsK when compared to mock-transfected cells. In TRAP-overexpressing cells colocalization of TRAP 5a and proCtsK was augmented, providing a plausible mechanism for generation of TRAP 5b. CtsK expression has been associated with cancer progression and has been pharmacologically targeted in several clinical studies.ResultsIn the current study, CtsK inhibition with MK-0822/Odanacatib did not abrogate the formation of TRAP 5b, but reversibly increased the intracellular levels of a N-terminal fragment of TRAP 5b and reduced secretion of TRAP 5a reversibly. However, MK-0822 treatment neither altered intracellular TRAP activity nor TRAP-dependent cell migration, suggesting involvement of additional proteases in proteolytic processing of TRAP 5a. Notwithstanding, CtsK was shown to be colocalized with TRAP and to be involved in the regulation of secretion of TRAP 5a in a breast cancer cell line, while it still was not essential for processing of TRAP 5a to TRAP 5b isoform.ConclusionIn cancer cells multiple proteases are involved in cleaving TRAP 5a to high-activity phosphatase TRAP 5b. However, CtsK-inhibiting treatment was able to reduce secretion TRAP 5a from TRAP-overexpressing cancer cells.
Highlights
Tartrate–resistant acid phosphatase (TRAP/ ACP5) belongs to the binuclear metallophosphatase family and is present in two isoforms
It was investigated if inhibition of Cathepsin K (CtsK) would inhibit proteolytic processing of Tartrate-resistant acid phosphatase (TRAP), and would this be sufficient to abrogate the functional effects of TRAP-overexpression, such as increased migration
In TRAP3high cell lysates TRAP 5b amounts were significantly increased, while the TRAP 5b isoform was absent in mock control cells (Fig. 1b), suggesting that the bulk of the overexpressed rat TRAP 5a is efficiently converted to the cleaved 5b isoform
Summary
Tartrate–resistant acid phosphatase (TRAP/ ACP5) belongs to the binuclear metallophosphatase family and is present in two isoforms. TRAP 5a can be post-translationally processed to a cleaved TRAP 5b isoform with high phosphatase activity by e.g. cysteine proteinases, such as Cathepsin K (CtsK). Tartrate-resistant acid phosphatase (TRAP/ACP5) is a metalloenzyme existing as two isoforms [1] It is synthesized as the uncleaved highly glycosylated 35 kDa molecule TRAP 5a, containing a domain constituting a loop of around 20 amino acids, which is restricting its Unlike e.g. serine proteases that typically have high selectivity towards specific substrates [6] TRAP is relatively. Its localization to vesicles and activation by proteolysis have been suggested as means to limit its function to a more specific set of substrates [7,8,9,10] Several proteinases, such as trypsin and cysteine proteinases have been assessed for their potential to cleave and activate TRAP 5a [4, 11]. TRAP 5a has been suggested to function as a growth factor for cells of the mesenchymal lineage [14]
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