The Importin‐Alpha/Nucleophosmin Switch Controls Taspase1 Protease Function

Abstract

Taspase1 is a threonine protease suspected to process (patho)biologically relevant nuclear and cytoplasmic substrates, such as the mixed lineage leukemia protein. However, neither the mechanisms regulating Taspase1's intracellular localization nor their functional consequences are known. Analysis of endogenous and ectopically expressed Taspase1 detected the protease predominantly in the nucleus accumulating at the nucleolus. Microinjection and ectopic expression studies identified an evolutionarily conserved bipartite nuclear import signal (NLS) (amino acids 197 K RNKRK LELA ERVDTDFMQLK KRR 220 ) interacting with importin-α. Notably, an NLS-mutated, import-deficient Taspase1 was biologically inactive. Although the NLS conferred nuclear transport already of the proenzyme, Taspase1's nucleolar localization required its autoproteolytic processing, triggering its interaction with the nucleolar shuttle protein nucleophosmin. In contrast, (auto)catalytically inactive Taspase1 mutants neither accumulated at the nucleolus nor bound nucleophosmin. Active nuclear import and interaction with nucleophosmin was found to be required for the formation of proteolytically active Taspase1 ensuring to efficiently process its nuclear targets. Intriguingly, coexpression of pathological nucleophosmin variants increased the amount of cytoplasmic Taspase1. Hence, Taspase1 appears to exploit the nuclear export activity of nucleophosmin to gain transient access to the cytoplasm required to also cleave its cytoplasmic substrates. Collectively, we here describe a hitherto unknown mechanism regulating the biological activity of this protease.

Overexpression of the Catalytically Impaired Taspase1T234V or Taspase1D233A Variants Does Not Have a Dominant Negative Effect in T(4;11) Leukemia Cells

“…[7], [23] Expression constructs encoding Taspase1 as a fusion with the small HA-tag was generated as described. [9], [24] The Taspase1 expression plasmid was used as template to amplify the Taspase1 α- and β-subunits.…”

“…pF143 encoding GFP, F145 encoding BFP and pBluescript (BSK) were described. [23], [26] Bacterial expression plasmids pGEX_GST-Tasp-GFP and pGEX_GST-GFP were described. [20] A detailed overview of plasmids and oligonucleotides used for PCR amplification and cloning can be found in Table S2 and S3 .…”

“…Leukemic and solid cancer cell lines used in the study were maintained and transfected as described [9], [23]. Observation and image analysis of living or fixed cells were performed as described [7], [27].…”

“…Preparation of whole lysates from cells and immunoblotting were carried out as described [23]. Equal loading of lysates was controlled by reprobing blots for GapDH as described [7].…”

“…To complete protein elution, 50 µl elution buffer were applied. 30 µl of the eluate, as well as 3% of input were analyzed as outlined [23].…”