Meccanismi di regolazione della chinasi HIPK2
Author(s)
Gatti, Veronica
Date Issued
June 19, 2014
Type
Doctoral Thesis
Abstract
HIPK2 is an evolutionarily conserved DYRK-like kinase originally identified for its capacity to interact
with homeodomain transcription factors. HIPK2 binds and phosphorylates a still enlarging body of
targets and is involved in the regulation of cell survival and proliferation during development and in
response to genotoxic damage or hypoxia. In this study, we focused our attention on two different
regulatory mechanisms of HIPK2 activity: phosphorylation and alternative splicing. To analyze HIPK2
phosphorylation pattern we worked with full-length protein.
Mass spectrometric analysis of GST-tagged wild-type HIPK2, produced and purified from
mammalian cells, allowed the identification of 22 phosphorylation sites spread throughout all protein
domains. Among these sites, we identified Y354 that belongs to an aminoacidic sequence conserved in
most protein kinases called activation loop. We demonstrated that activation-loop Y phosphorylation is
an autocatalytic event important for kinase activity achievement, substrate specificity and correct
cellular localization. Indeed, inhibition of activation-loop Y phosphorylation induces a strong decrease
in the phosphorylation of the two specific substrates, p53 and p63 and the acquisition of aberrant Y and
S/T kinase activity. Out-of-target phosphorylation causes a strong cytoplasmic delocalization together
with protein aggregates formation resembling the observation made in different tumors by
immunohistochemistry.
Although the existence of HIPK2 splicing isoforms has been hypothesized, it has not
systematically verified thus far. In this work we showed that HIPK2 human gene is subjected to
alternative splicing regulation and that it encodes, besides full-length mRNA, at least two more isoforms.
The first alternative splicing consists in the alternative 3’-splice site selection in exon 8 (Δe8) causing
the loss of 81 bps and 27 aminoacids encoding a portion of homeodomain interacting domain; the
second alternative splicing consists in the retention of intron13 (i13). Because of a stop codon after 89
bps of the intron, HIPK2i13 protein loses its C-terminal part and gains 29 new aminoacids. We showed
that mRNA levels of each isoform are strictly dependent on the amount of other isoforms messengers,
suggesting a complex regulation. A further layer of complexity, due to different post-translational
modifications, strongly contributes to this variability. Indeed, Western blot experiments on nuclear and
cytoplasmic extracts suggest the existence of at least two different forms of full-length protein; one of
them has such a post-translational modification pattern that it localize preferentially in the nucleus and
the other one exclusively in the cytoplasm. Moreover, we observed a Δe8 isoform with an exclusively
nuclear localization in normal cells that is partially delocalized in the cytoplasm in tumor cells.
Preliminary functional experiments suggest that HIPK2 splicing isoforms could have different functions,
as described for other tumor suppressor. In particular, we showed that i13 has the strongest cell death
activity.
Additional information
Dottorato di ricerca in Genetica e biologia cellulare
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