Death Associated Protein Kinase (DAPK) has a wide-ranging role in cell death
signaling and growth control. Over the past decade the importance of DAPK as a
tumour suppressor has been highlighted by numerous studies that show its expression
is ablated in many cancer types by epigenetic silencing. However the mechanisms by
which this multi-domain protein exerts death-inducing effects have not been well
defined, given that very few substrates or interaction partners have been discovered.
Many protein-protein interactions involving cell signaling processes are driven by
linear interaction motifs. Therefore combinatorial peptide libraries displayed on Ml3
filamentous bacteriophage were used to identify peptide consensus binding sites for
the kinase domain of DAPK. Peptides that bound to the DAPKcore kinase domain
were then isolated and sequenced leading to the discovery of binding peptides with
striking homology to the SHI-4 family of transcription factors, the Promyelocytic
Leukemia protein (PML) and the microtubule associating protein MAP IB.
Immunobinding assays, immunofluorescent cell staining studies and biochemical
fractionations demonstrated that DAPK can interact with human MAP IB via an Nterminal interface in-vitro and in cells and so this interaction was subject to further
study. DAPK has been shown to integrate death inducing signals through a number
of pathways including the p53 tumour suppression pathway and apoptotic and
autophagic cell death inducing pathways. Therefore a range of assays were
developed to characterise the biological significance of DAPK interaction with
MAP IB in the context of each pathway. Cell growth and viability assays
demonstrated that MAP IB co-operates with DAPK to reduce cell proliferation. This
co-operative cell growth inhibition was independent of the p53 pathway and
apoptotic (Type I) cell death, but induced autophagic (Type II) cell death. MAP IB
co-operation with DAPK was marked by a striking increase in the number of cells
with membrane blebbing morphology, an effect previously shown to involve DAPK
interaction with the actin cytoskeleton leading to actin-myosin contraction. This was
in contrast to the known role of MAP IB that is primarily thought of as a tubulin
associating protein that modifies microtubule dynamics. Therefore the role of the
cytoskeleton in DAPK co-operation with MAP IB was studied in detail using
immunofluorescent cytoskeleton staining and microtubule purification assays.
During DAPK transfection induced membrane blebbing, a pool of DAPK and
MAP IB co-localise and co-purify with tubulin whereas a separate pool is co-located
to cortical actin. Thus DAPK and MAP IB cooperation-induced membrane blebbing
involves a novel interaction with both microtubules and microfilaments. These
studies highlight the utility of peptide combinatorial libraries to identify novel
binding interfaces and highlight a positive role for MAP IB in DAPK dependent
cytoskeletal rearrangement and the autophagic cell death program
