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. 2000 Feb 15;97(4):1572-7.
doi: 10.1073/pnas.020519497.

A functional genetic screen identifies regions at the C-terminal tail and death-domain of death-associated protein kinase that are critical for its proapoptotic activity

T Raveh  1 , H Berissi, M Eisenstein, T Spivak, A Kimchi
Affiliations

A functional genetic screen identifies regions at the C-terminal tail and death-domain of death-associated protein kinase that are critical for its proapoptotic activity

T Raveh et al. Proc Natl Acad Sci U S A. .

Abstract

Death-associated protein kinase (DAP-kinase) is a Ca(+2)/calmodulin-regulated serine/threonine kinase with a multidomain structure that participates in apoptosis induced by a variety of signals. To identify regions in this protein that are critical for its proapoptotic activity, we performed a genetic screen on the basis of functional selection of short DAP-kinase-derived fragments that could protect cells from apoptosis by acting in a dominant-negative manner. We expressed a library of randomly fragmented DAP-kinase cDNA in HeLa cells and treated these cells with IFN-gamma to induce apoptosis. Functional cDNA fragments were recovered from cells that survived the selection, and those in the sense orientation were examined further in a secondary screen for their ability to protect cells from DAP-kinase-dependent tumor necrosis factor-alpha-induced apoptosis. We isolated four biologically active peptides that mapped to the ankyrin repeats, the "linker" region, the death domain, and the C-terminal tail of DAP-kinase. Molecular modeling of the complete death domain provided a structural basis for the function of the death-domain-derived fragment by suggesting that the protective fragment constitutes a distinct substructure. The last fragment, spanning the C-terminal serine-rich tail, defined a new regulatory region. Ectopic expression of the tail peptide (17 amino acids) inhibited the function of DAP-kinase, whereas removal of this region from the complete protein caused enhancement of the killing activity, indicating that the C-terminal tail normally plays a negative regulatory role. Altogether, this unbiased screen highlighted functionally important regions in the protein and revealed an additional level of regulation of DAP-kinase apoptotic function that does not affect the catalytic activity.

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Figures

Figure 1
Figure 1
Isolation of DAP-kinase fragments that confer resistance to IFN-γ-induced apoptosis. (A) Screening strategy. Purified human DAP-kinase cDNA underwent partial DNaseI digestion, and fragments were subcloned into an EBV-derived expression vector to generate a cDNA library of random fragments. This library was introduced into HeLa cells, and elements that conferred resistance to apoptosis were isolated and further analyzed. (B) Vector design for the DAP-kinase-fragmented cDNA library. pTKO1, an EBV derived vector, was modified to accommodate the cDNA library by insertion of the indicated adaptor (for details, see Materials and Methods).
Figure 2
Figure 2
Protective fragments of DAP-kinase isolated by the genetic screen. Biologically active fragments that passed the two successive screens are listed in the table (Upper). (Lower) Schematic representation of DAP-kinase full-length protein with the position of the library-derived cell death-protective fragments shown underneath.
Figure 3
Figure 3
DAP-kinase-derived fragments protect cells from apoptosis. (A) Apoptosis was induced in 293 cells by transient overexpression of p55 TNF-receptor. The receptor was expressed together with an empty vector, a dominant-negative mutant of FADD, or the different DAP-kinase fragments, as indicated. Transfected cells were identified by GFP expression, and the rate of cell death was scored according to typical morphological features. The graph represents average values obtained from three independent experiments, each of which included at least 300 GFP-positive cells. (B) 293 cells were induced to undergo apoptosis by transient overexpression of activated DAP-kinase (Δ-CaM). The cells were transfected with a plasmid encoding this mutant, together with either an empty vector or different fragments, as indicated. Transfectants were identified by GFP expression, and apoptosis was scored as in A. Below is an immunoblot containing equal amounts of total cell extracts, which were reacted with anti-DAP-kinase antibodies to compare the levels of exogenous DAP-kinase in the different transfections (the endogenous levels are below detection levels under these exposure conditions). (C) MCF7 cells were transfected with p55 TNF-receptor together with an empty vector, the same vector containing the FADD death domain or different DAP-kinase derived fragments, as indicated. Apoptosis was scored as in A.
Figure 4
Figure 4
Model of the death domain of DAP-kinase. (A) Sequence alignment of the death domains of DAP-kinase (amino acids 1300–1398) and p75 neurotrophin receptor (amino acids 334–420). Amino acids that are included in the protective fragment (1320–1371) are marked by blue letters. Amino acids that form the six α-helical structures are emphasized in bold letters and brackets. (B) Model structure of the death domain of DAP-kinase (colored ribbons) constructed by comparative modeling and overlaid on the NMR-based structure of the p75 neurotrophin receptor (white ribbon). The six helices (α1 to α6) are accentuated by yellow cylinders. In the DAP-kinase death-domain model, regions that are inside and outside the protective fragment are marked with blue and purple, respectively. Note the extended loops between helices α1 and α2, and α3 and α4 in DAP-kinase compared with p75. (C) Model structure of the death domain of DAP-kinase presented as space-filling spheres. As in B, the protective fragment is colored blue.
Figure 5
Figure 5
Deletion of the last 17 amino acids of DAP-kinase potentiates its activity. (A) 293 cells were transfected with either an empty vector or the same vector carrying the indicated forms of DAP-kinase. WT, wild-type protein; Δ-tail, a truncated mutant lacking the 17 C-terminal amino acids; ΔCaM, a deletion mutant lacking the CaM regulatory region. Transfected cells were identified by GFP coexpression, and the rate of cell death was assessed 24 hr posttransfection, according to typical apoptotic morphology. The graph represents values from three independent experiments, each including at least 300 GFP-positive cells. (B) Protein extracts were prepared from the transfected 293 cells (see A), and DAP-kinase protein levels were analyzed by Western blotting by using specific monoclonal antibodies. The endogenous DAP-kinase was below detection levels under these exposure conditions. (C) An in vitro kinase assay was performed with proteins immunoprecipitated from 293 cells transfected with the indicated plasmids. The assay included an exogenous substrate, MLC, and the relative activity was determined according to the rate of MLC phosphorylation, as quantified by using a PhosphorImager.
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