Synthesis and phorbol ester-binding studies of the individual cysteine-rich motifs of protein Kinase D

doi:10.1016/S0960-894X(99)00413-8

Bioorganic & Medicinal Chemistry Letters

Volume 9, Issue 17, 6 September 1999, Pages 2487-2490

https://doi.org/10.1016/S0960-894X(99)00413-8 Get rights and content

Abstract

To investigate the phorbol ester-binding properties of the individual cysteine-rich motifs of protein kinase D (PKD), the 52-mer peptides containing each cysteine-rich motif of PKD (PKD-C1A, PKD-C1B) have been synthesized. The [³H]phorbol-12,13-dibutyrate (PDBu) binding to PKD-C1A was affected drastically by incubation temperature while that to PKD-C1B was not. Scatchard analysis of [³H]PDBu binding to both PKD C1 peptides gave dissociation constants of 2.5 ± 0.4 and 2.7 ± 0.8 nM for PKD-C1A and PKD-C1B, respectively, indicating that the two cysteine-rich motifs of PKD are functionally equivalent like those of PKCγ.

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Potein kinase D signaling in cancer: A friend or foe?
2017, Biochimica et Biophysica Acta - Reviews on Cancer
Citation Excerpt :
Overall, C1 domain is central to the spatial and temporal regulation of PKD localization at different subcellular locations. In addition to the intrinsic differences of the twin C1 domains [20,24,25], their ligand binding activity, selectivity, and accessibility to ligand in a holoenyme are intricately regulated by PKD phosphorylation [22], kinase activity [19,21,22] and interactions with protein binding partners [26], and this regulation becomes more complex with embedded nuclear localization and export signals within the structure of C1 domain [27]. Whether, how, and how much they contribute to the differential biological functions of PKD isoforms remain to be determined.
Protein kinase D is a family of evolutionarily conserved serine/threonine kinases that belongs to the Ca⁺⁺/Calmodulin-dependent kinase superfamily. Signal transduction pathways mediated by PKD can be triggered by a variety of stimuli including G protein-coupled receptor agonists, growth factors, hormones, and cellular stresses. The regulatory mechanisms and physiological roles of PKD have been well documented including cell proliferation, survival, migration, angiogenesis, regulation of gene expression, and protein/membrane trafficking. However, its precise roles in disease progression, especially in cancer, remain elusive. A plethora of studies documented the cell- and tissue-specific expressions and functions of PKD in various cancer-associated biological processes, while the causes of the differential effects of PKD have not been thoroughly investigated. In this review, we have discussed the structural-functional properties, activation mechanisms, signaling pathways and physiological functions of PKD in the context of human cancer. Additionally, we have provided a comprehensive review of the reported tumor promoting or tumor suppressive functions of PKD in several major cancer types and discussed the discrepancies that have been raised on PKD as a major regulator of malignant transformation.
Individual C1 domains of PKD3 in phorbol ester-induced plasma membrane translocation of PKD3 in intact cells
2005, Cellular Signalling
Protein kinase D3 is a novel member of the serine/threonine kinase family PKD. The regulatory region of PKD contains a tandem repeat of C1 domains designated C1a and C1b that bind diacylglycerol and phorbol esters, and are important membrane targeting modules. Here, we investigate the activities of individual C1 domains of PKD3 and their roles in phorbol ester-induced plasma membrane translocation of PKD3. Truncated C1a of PKD3 binds [³H]phorbol 12, 13-dibutyrate with high affinity, but no binding activity is detected for C1b. Meanwhile, mutations in C1a of truncated C1ab of PKD3 lead to the loss of binding affinity, while these mutations in C1b have little impact, indicating that C1a is responsible for most of the phorbol ester-binding activities of PKD3. C1a and C1b of the GFP-tagged full length PKD3 are then mutated to assess their roles in phorbol ester-induced plasma membrane translocation in intact cells. At low concentration of phorbol 12-myristate 13-acetate (PMA), the plasma membrane translocations of the C1a and C1ab mutants are significantly impaired, reflecting an important role of C1a in this process. However, at higher PMA concentrations, all C1 mutants exhibit increased rates of translocation as compared to that of wild-type PKD3, which parallel their enhanced activation by PMA, implying that PKD3 kinase activity affects membrane targeting. In line with this, a constitutive active PKD3-GFP translocates similarly as wild-type PKD3, while a kinase-inactive PKD3 shows little translocation up to 2 μM PMA. In addition, RO 31-8220, a potent PKC inhibitor that blocks PMA-induced PKD3 activation in vivo, significantly attenuates the plasma membrane translocation of wild-type PKD3 at different doses of PMA. Taken together, our results indicate that both C1a and the kinase activity of PKD3 are necessary for the phorbol ester-induced plasma membrane translocation of PKD3. PKC, by directly activating PKD3, regulates its plasma membrane localization in intact cells.
Synthesis and phorbol ester binding of the cysteine-rich domains of diacylglycerol kinase (DGK) isozymes. DGKγ and DGKβ are new targets of tumor-promoting phorbol esters
2003, Journal of Biological Chemistry
Citation Excerpt :
These peptides were folded using zinc chloride by a method reported previously (23, 24) and subjected to a PDBu binding assay. Because our recent investigation found that some C1 domain fragments of PKC isozymes suffered from temperature-dependent inactivation (24, 33), the incubation temperature of the binding assay was set at 4 °C for the DGK isozyme C1 peptides. The specific binding to PDBu of the DGK C1 peptides is summarized in Fig.3.
Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGKγ-C1A and DGKβ-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGKγ-C1A, human-DGKγ-C1A, and human-DGKβ-C1A gaveK_d values of 3.6, 2.8, and 14.6 nm, respectively, suggesting that DGKγ and DGKβ are new targets of phorbol esters. An A12T mutation of human-DGKβ-C1A enhanced the affinity to bind PDBu, indicating that the β-hydroxyl group of Thr-12 significantly contributes to the binding. TheK_d value for PDBu of FLAG-tagged whole rat-DGKγ (4.4 nm) was nearly equal to that of rat-DGKγ-C1A (3.6 nm). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGKγ and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGKγ to cause its translocation.
Mechanism of persistent protein kinase D1 translocation and activation
2003, Developmental Cell
The specificity of many signal transduction pathways relies on the spatiotemporal features of each signaling step. G protein-coupled receptor-mediated activation of protein kinases leads to diverse cellular effects. Upon receptor activation, PKD1 and several C-type protein kinases (PKCs), translocate to the plasma membrane and become catalytically active. Here we show that, unlike PKCs, PKD1 remains active at the membrane for hours. The two DAG binding C1 domains of PKD1 have distinct functional roles in targeting and maintaining PKD1 at the plasma membrane. C1A achieves fast, maximal, and reversible translocation, while C1B translocates partially, but persistently, to the plasma membrane. The persistent localization requires the C1B domain of PKD1, which binds Gαq. We incorporate the kinetics of PKD1 translocation into a three-state model that suggests how PKD1 binding to DAG and Gαq uniquely encodes frequency-dependent PKD1 signaling.
Establishment of a binding assay for protein kinase C isozymes using synthetic C1 peptides and development of new medicinal leads with protein kinase C isozyme and C1 domain selectivity
2002, Pharmacology and Therapeutics
Conventional and novel protein kinase C (PKC) isozymes contain two cysteine-rich C1 domains (C1A and C1B), both of which are candidate phorbol-12, 13-dibutyrate (PDBu)-binding sites. We synthesized C1 peptides of 50–70 residues corresponding to all PKC isozyme C1 domains using an Fmoc solid-phase strategy. These C1 peptides were successfully folded by zinc treatment, as monitored by electrospray ionization time-of-flight mass spectrometry. We measured the K_d's of [³H]PDBu for all PKC C1 peptides. Most of the C1 peptides, except for δ-C1A and θ-C1A, showed strong PDBu binding affinities with K_d's in the nanomolar range (0.45–7.4 nM) comparable with the respective whole PKC isozymes. The resultant C1 peptide library can be used to screen for new ligands with PKC isozyme and C1 domain selectivity. Non-tumor-promoting 1-oleoyl-2-acetyl-sn-glycerol and bryostatin 1 showed relatively strong binding to all CIA peptides of novel PKCs (δ, ε, and η). In contrast, the tumor promoters (−)-indolactam-V, ingenol-3-benzoate, and PDBu bound selectively to all C1B peptides of novel PKCs. The preference of tumor promoters for the domain might be related to tumorigenesis since recent investigations proposed the involvement of novel PKCs in tumor promotion in vivo using transgenic or knockout mice. Moreover, we recently have found that a new lactone analogue of benzolactams (6) shows significant selectivity in PKCη-C1B binding.
Diacylglycerol kinase γ is one of the specific receptors of tumor-promoting phorbol esters
2001, Biochemical and Biophysical Research Communications
Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two different enzyme families that interact with diacylglycerol. Both enzymes contain cysteine-rich C1 domains with a zinc finger-like structure. Most of the C1 domains of PKCs show strong phorbol-12,13-dibutyrate (PDBu) binding with nanomolar dissociation constants (K_d's). However, there has been no experimental evidence that phorbol esters bind to the C1 domains of DGKs. We focused on DGKγ because its C1A domain has a high degree of sequence homology to those of PKCs, and because DGKγ translocates from the cytoplasm to the plasma membrane following 12-O-tetradecanoylphorbol-13-acetate treatment similar to PKCs. Two C1 domains of DGKγ (DGKγ-C1A and DGKγ-C1B) were synthesized and tested for their PDBu binding along with whole DGKγ (Flag-DGKγ) expressed in COS-7 cells. DGKγ-C1A and Flag-DGKγ showed strong PDBu binding affinity, while DGKγ-C1B was completely inactive. Scatchard analysis of DGKγ-C1A and Flag-DGKγ gave K_d's of 3.1 and 4.4 nM, respectively, indicating that the major PDBu binding site of DGKγ is C1A. This is the first evidence that DGKγ is a specific receptor of tumor-promoting phorbol esters.

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Synthesis and phorbol ester-binding studies of the individual cysteine-rich motifs of protein Kinase D

Abstract

J. Biol. Chem.

J. Biol. Chem.

Tetrahedron Lett.

Anal. Biochem.

Cell

FEBS Lett.

J. Biol. Chem.

FEBS Lett.

Protein Sci.

Enzyme Protein

FASEB J.