A Death-associated Protein Kinase (DAPK)-interacting Protein, DIP-1, Is an E3 Ubiquitin Ligase That Promotes Tumor Necrosis Factor-induced Apoptosis and Regulates the Cellular Levels of DAPK

Abstract

Death-associated protein kinase (DAPK) is a multidomain Ser/Thr protein kinase with an important role in apoptosis regulation. In these studies we have identified a DAPK-interacting protein called DIP-1, which is a novel multi-RING finger protein. The RING finger motifs of DIP-1 have E3 ligase activity that can auto-ubiquitinate DIP-1 in vitro. In vivo, DIP-1 is detected as a polyubiquitinated protein, suggesting that the intracellular levels of DIP-1 are regulated by the ubiquitin-proteasome system. Transient expression of DIP-1 in HeLa cells antagonizes the anti-apoptotic function of DAPK to promote a caspase-dependent apoptosis. These studies also demonstrate that DAPK is an in vitro and in vivo target for ubiquitination by DIP-1, thereby providing a mechanism by which DAPK activities can be regulated through proteasomal degradation.Regulation of protein degradation by the ubiquitin proteasome pathway is now known to be a major pathway through which cells modulate the expression levels of critical signaling proteins (1-6). This tightly regulated, complex pathway is a key regulator of many important signaling pathways and has an important role in many cellular processes including apoptosis, and recent studies have identified many apoptosis regulatory proteins as targets for ubiquitination (7-11). In addition to being targets for degradation, some apoptosis regulatory proteins have a more active role and act as components of the ubiquitin cascade via the ubiquitin ligase activity ascribed to the RING finger domains that is part of their primary structure. Targeting proteins for degradation by the ubiquitin proteasome pathway involves the covalent linkage of ubiquitin either to the amino terminus or specific lysine residues in the target protein through the action of three enzymes. In this process ubiquitin is first activated by an E1 ubiquitin-activating enzyme, transferred to an E2 ubiquitin-conjugating enzyme, and then ligated to the target protein by an E3 ubiquitin ligase (4,12) Recently the Ser/Thr protein kinase, death-associated protein kinase (DAPK) 1 has been implicated in apoptosis regulation. DAPK has a complex, multi-domain structure that includes a calcium/calmodulin-regulated kinase domain, a series of ankyrin repeats, and a carboxyl-terminal death domain (13-17). Although some of the regulatory features that directly control the catalytic activities of DAPK have been described, including the activation by calcium/calmodulin (17, 18) and the presence of an inhibitory autophosphorylation site (19), an understanding of how the cellular activities of DAPK are regulated in vivo is poorly understood. The presence of proteinprotein interaction domains within the primary structure of DAPK, including its ankyrin repeat motifs and death domain, suggests that additional interactions between DAPK and other cellular proteins will also be important for regulation of DAPK activities. In this study, we describe a new DAPK-interacting protein called DIP-1 (DAPK-interacting protein-1), which has a direct role in r...

A ubiquitin ligase, skeletrophin, is a negative regulator of melanoma invasion

“…Skeletrophin, and its paralogue, mind bomb (also reported as DIP-1 (Jin et al, 2002) or mind bomb-1 (Koo et al, 2005b), target the intracellular region of Notch ligands and mediates ligand-dependent Notch activation. Internalization of Notch ligands followed by ubiquitination by skeletrophin and mind bomb is critical for Notch signal transduction (Itoh et al, 2003;Koo et al, 2005a, b;Pitsouli and Delidakis, 2005;Takeuchi et al, 2005;Wang and Struhl, 2005).…”

“…Skeletrophin and its paralogue, mind bomb (the former is also reported as mindbomb homolog 2 (MIB2), and the latter as DIP-1 (DAPK (deathassociated protein kinase)-interacting protein) (Jin et al, 2002) or mind bomb-1) mediate ligand-dependent Notch activation by ubiquitination of the intracellular region of Notch ligands in a RING-finger domain-dependent manner. These findings indicate that the silencing of skeletrophin expression may be related to aberrant Notch signal transduction in melanoma cells.…”

HYPERSENSITIVE TO RED AND BLUE 1, a ZZ-Type Zinc Finger Protein, Regulates Phytochrome B–Mediated Red and Cryptochrome-Mediated Blue Light Responses

“…Within the CREB binding and p300 proteins, the binding sites for E1A and TFIIB have been narrowed down to regions that contain the ZZ-type zinc finger and a second TAZ zinc finger (Davies et al, 1996). The motifs in RING domain proteins may function as additional sites of protein-protein interactions (Jin et al, 2002;Itoh et al, 2003). We infer that ZZ-type zinc finger domain in HRB1 is likely involved in interactions with other light signaling components in either the red or blue light signaling pathway.…”

“…The motif is also present in many RING domain proteins thought to be involved in protein traffic or protein degradation (Jin et al, 2002;Itoh et al, 2003). The ZZ-type zinc finger motif in dystrophin appears to bind calmodulin, and a missense mutation on one of the conserved Cys residues in dystrophin causes Duchenne muscular dystrophy (Davies et al, 1996).…”

Quantitative and correlation analysis of the DNA methylation and expression of DAPK in breast cancer

“…The protein expression of DAPK is controlled by both ubiquitin-proteasome (Jin et al, 2002;Lee et al, 2010;Zhang, Nephew, & Gallagher, 2007) and lysosome pathways (Gallagher & Blue, 2014;Lin et al, 2011;Lin et al, 2009;Lin et al, 2008;Stevens et al, 2009). It is not surprising that the mRNA and protein expression of DAPK did not correlate well (Figure 3 …”