DIK, a Novel Protein Kinase That Interacts with Protein Kinase Cδ

Abstract

A novel serine/threonine kinase, termed DIK, was cloned using the yeast two-hybrid system to screen a cDNA library from the human keratinocyte cell line HaCaT with the catalytic domain of rat protein kinase C␦ (PKC␦ cat ) cDNA as bait. The predicted 784-amino acid polypeptide with a calculated molecular mass of 86 kDa contains a catalytic kinase domain and a putative regulatory domain with ankyrin-like repeats and a nuclear localization signal. Expression of DIK at the mRNA and protein level could be demonstrated in several cell lines. The dik gene is located on chromosome 21q22.3 and possesses 8 exons and 7 introns. DIK was synthesized in an in vitro transcription/translation system and expressed as recombinant protein in bacteria, HEK, COS-7, and baculovirus-infected insect cells. In the in vitro system and in cells, but not in bacteria, various post-translationally modified forms of DIK were produced. DIK was shown to exhibit protein kinase activity toward autophosphorylation and substrate phosphorylation. The interaction of PKC␦ cat and PKC␦ with DIK was confirmed by coimmunoprecipitation of the proteins from HEK cells transiently transfected with PKC␦ cat or PKC␦ and DIK expression constructs.The members of the PKC 1 family, because of structural and enzymatic differences, can be subdivided into several groups (for reviews see Refs. 1 and 2). PKC␦, a member of the so-called nPKC subfamily, has attracted the interest of an increasing number of research groups over the last years and presumably is one of the most thoroughly studied PKC isoenzymes (for a review see Ref. 3). Like all the other PKC isoforms, PKC␦ is thought to play an individual role in various signaling pathways and to specifically affect diverse cellular processes, such as growth, differentiation, apoptosis, and tumorigenesis (4 -16). This specific action is likely to afford a sophisticated network of regulation of PKC␦ activity, subcellular localization, and substrate phosphorylation. Beside the well known regulation of enzyme activity by signal-induced second messengers, such as diacylglycerol, PKC␦ is regulated by up-and downmodulation of its expression (4, 17, 18), by phosphorylation (19 -29), and presumably by interaction with other proteins involved in signal transduction, such as other protein kinases and anchor or docking proteins (30 -38). Particularly the latter is essential for a specific subcellular localization of the enzyme and a selective phosphorylation of substrate proteins. Our knowledge of PKC␦-protein interactions, however, is rather scanty. This holds true not only for the interaction with physiologically relevant substrate proteins but also for the interaction with other proteins that might affect PKC␦ signaling.We therefore attempted to clone proteins interacting with PKC␦ by using the yeast two-hybrid system (YTHS). Here, we describe the cloning, expression, and characterization of a novel serine/threonine kinase, termed DIK (PKC-delta-interacting protein kinase), which is coimmunoprecipitated with PKC␦ and the catalyti...

Receptor-interacting Protein Kinase 4 and Interferon Regulatory Factor 6 Function as a Signaling Axis to Regulate Keratinocyte Differentiation

“…However, its delayed induction in response to PMA suggests that IRF6 indirectly regulates MAD1 expression. PKC␦, which has been shown previously to form a complex with RIPK4 (11), is an important regulator of keratinocyte differentiation (45)(46)(47). For example, PKC␦ promotes IVL gene expression by up-regulating the expression of the transcription factor Kruppel-like factor 4 (KLF4) (47).…”

RIP kinases: key decision makers in cell death and innate immunity

“…The functions of RIP 4-7 are poorly understood and are well reviewed elsewhere. 7 Briefly, RIP4 was initially identified as a PKCd-interacting protein 8 and was subsequently shown to activate NFkB. 9 It has a key role in keratinocyte differentiation 10 and cutaneous inflammation.…”

Protein Kinase C-associated Kinase (PKK) Mediates Bcl10-independent NF-κB Activation Induced by Phorbol Ester

“…It has been hypothesized that PKK and its human orthologue are somehow involved in a PKC-associated signaling pathway (14,15). However, the particular signaling pathway in which PKK functions has not been previously addressed.…”

“…These observations suggest that proteins carrying kinase domains homologous to those of RIP and RICK, but different C-terminal domains, might be involved in the activation of novel NF-B signaling pathways. PKK, a mouse kinase composed of an N-terminal kinase domain, an IM region, and a C-terminal domain containing 11 ankyrin repeats was recently identified for its ability to interact with protein kinase C (PKC) isoform PKC␤I, whereas its human counterpart named DIK was shown to associate with PKC␦ (14,15). PKCs mediate intracellular signals triggered by stimulation of a variety of extracellular ligands including those associated with G-coupled and antigen receptors (16).…”