Franz-Josef Johannes

Inhibition of protein kinase C μ by various inhibitors. Inhibition from protein kinase c isoenzymes

Various inhibitors were tested for their potential to suppress the kinase activity of protein kinase C μ (PKCμ) in vitro and in vivo. Among the staurosporine‐derived, rather selective PKC inhibitors the indolocarbazole Gö 6976 previously shown to inhibit preferentially cPKC isotypes proved to be a potent inhibitor of PKCμ with an IC5 of 20 nM, whereas the bisindolymaleimide Gö 6983 was extremely ineffective in suppressing PKCμ kinase activity with a thousand‐fold higher ICm of 20 μM. Other strong inhibitors of PKCμ were the rather unspecific inhibitors staurosporine and K252a. Contrary to the poor inhibition of PKCμ by Gö 6983, this compound was found to suppress in vitro kinase activity of PKC isoenzymes from all three subgroups very effectively with IC50 values from 7 to 60 nM. Thus, Gö 6983 was able to differentiate between PKCμ and other PKC isoenzymes being useful for selective determination of PKCμ kinase activity in the presence of other PKC isoenzymes.

Determination of the Specific Substrate Sequence Motifs of Protein Kinase C Isozymes

Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (α, βI, βII, γ, δ, ε, η, μ, and ζ) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position +1 carboxyl-terminal of the phosphorylated Ser and basic residues at position −3. All isozymes, except PKCμ, selected peptides with basic amino acids at positions −6, −4, and −2. PKCα, -βI, -βII, -γ, and -η selected peptides with basic amino acid at positions +2, +3, and +4, but PKCδ, -ε, -ζ, and -μ preferred peptides with hydrophobic amino acid at these positions. At position −5, the selectivity was quite different among the various isozymes; PKCα, -γ, and -δ selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKCμ showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position −3 to +2 for PKCα, -βI, -βII, -γ, -δ, and -η were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKCα, -βI, -δ, -ζ, and -μ were prepared and used for the determination of Km and Vmax for these isozymes. As judged by Vmax/Km values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft.

Dominant-negative FADD inhibits TNFR60-, Fas/Apo1- and TRAIL-R/Apo2-mediated cell death but not gene induction.

Fas/Apo1 and other cytotoxic receptors of the tumor necrosis factor receptor (TNFR) family contain a cytoplasmic death domain (DD) [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] that activates the apoptotic process by interacting with the DD-containing adaptor proteins TNFR-associated DD protein (TRADD) [12] [13] and Fas-associated DD protein (FADD/MORT1) [14] [15], leading to the activation of cysteine proteases of the caspase family [16]. Stimulation of Fas/Apo1 leads to the formation of a receptor-bound death-inducing signaling complex (DISC), consisting of FADD and two different forms of caspase-8 [17] [18] [19]. Transient expression of a dominant-negative mutant of FADD impairs TNFR60-mediated and Fas/Apo1-mediated apoptosis [13] [20], but has no effect on TNF-related apoptosis-inducing ligand (TRAIL/Apo2L)-induced cell death [7] [8] [9] [10] [21]. To study the function of FADD in DD-receptor signaling in more detail, we established HeLa cells that stably expressed a green fluorescent protein (GFP)-tagged dominant-negative mutant of FADD, GFP-DeltaFADD. Interestingly, expression of this mutant inhibited cell death induced by TNFR60, Fas/Apo1 and TRAIL-R/Apo2. In addition, GFP-DeltaFADD did not interfere with TNF-mediated gene induction or with activation of NF-kappaB or Jun N-terminal kinase (JNK), demonstrating that FADD is part of the TNFR60-initiated apoptotic pathway but does not play a role in TNFR60-mediated gene induction. Fas/Apo1-mediated activation of JNK was unaffected by the expression of GFP-DeltaFADD, suggesting that in Fas/Apo1 signaling the apoptotic pathway and the activation of JNK diverge at a level proximal to the receptor, upstream of or parallel to FADD.

Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

Protein kinase Cμ (PKCμ), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952–960), and its mechanism of regulation is not yet clear. Here we show that PKCμ forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCμ between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCμ failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCμ-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCμ including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCμ acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase Cμ

To study enzymatic activity and activation conditions of the recently identified novel protein kinase C μ (PKCμ) subtype, epitope tagged PKCμ was propagated in the baculovirus expression system and was purified to homogeneity. PKCμ displays high affinity phorbol ester binding (K d = 7 nM) resulting in enhanced phosphatidylserine‐dependent kinase activity. From various lipid second messengers known to activate PKCs only diacylglycerol and PtdIns‐4,5‐P2, were found to promote PKCμ kinase activity. Two peptides derived from the glycogen synthase, GS‐peptide and syntide 2, were found to be phosphorylated efficiently in vitro. MARCKS (myristoylated alanine‐rich C‐kinase substrate) served as an in vitro substrate for PKCμ too. However, in contrast to other PKCs, a peptide derived from the MARCKS phosphorylation domain is phosphorylated only at serine 156, and not at serines 152 and 163, implicating a differential regulation by PKCμ.

Protein-kinase-Cμ expression correlates with enhanced keratinocyte proliferation in normal and neoplastic mouse epidermis and in cell culture

In order to gain insight into the biological function of a PKC iso-enzyme, the protein kinase Cμ, we analyzed the expression pattern of this protein in mouse epidermis and keratinocytes in culture. Daily analysis of neonatal mouse epidermis immediately after birth showed a time-dependent reduction in the PKCμ content. Expression of the proliferating-cell nuclear antigen (PCNA), indicative of the proliferative state of cells, was reduced synchronously with PKCμ as the hyperplastic state of the neonatal tissue declined. In epidermal mouse keratinocytes, fractionated according to their maturation state, PKCμ expression was restricted to PCNA-positive basal-cell fractions. In primary cultures of those cells, growth arrest and induction of terminal differentiation by Ca2+ resulted in strongly reduced PKCμ expression, concomitantly with the loss of PCNA expression. Treatment of PMK-R1 keratinocytes with 100 nM of the mitogen 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in activation of PKCμ, reflected by translocation from the cytosolic to the particulate fraction and by shifts in electrophoretic mobility. DNA synthesis was significantly inhibited by the PKCμ inhibitor Goedecke 6976, while Goedecke 6983 did not inhibit PKCμ. Carcinomas generated according to the 2-stage carcinogenesis protocol in mouse skin consistently exhibited high levels of PKCμ. These data correlate PKCμ expression with the proliferative state of murine keratinocytes and point to a role of PKCμ in growth stimulation. A correlation between PKCμ expression and enhanced cell proliferation was also observed for NIH3T3 fibroblasts transfected with and over-expressing human PKCμ. Int. J. Cancer 80:98–103, 1999.

Immunological Demonstration of Protein Kinase Cμ in Murine Tissues and Various Cell Lines

13 murine tissues and 12 cell lines were tested for the expression of the novel protein kinase C (PKC) isoenzyme mu. Using two different PKC mu antibodies (sc-639 and P26720), PKC mu was detected in all tissues and cells and thus proved to be an ubiquitous PKC isotype. However, in some tissues, PKC mu was recognized only by the antibody P26720 and not by sc-639. Thymus, lung and peripheral blood mononuclear cells expressed the greatest amount of PKC mu. Recognition of PKC mu by the antibody sc-639 was drastically impaired when treating keratinocytes or mouse skin in vivo with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), thus mimicking down-regulation of PKC mu. The lack of a decrease in the PKC mu amount and, thus, the lack of down-regulation could be proved using the antibody P26720. This antibody was able to recognize PKC mu in extracts of untreated as well as TPA-treated tissues or cells. Phosphorylation of proteins in a cell-free system (cell or tissue extracts) in the presence and absence of TPA or other PKC activators and various protein kinase inhibitors indicated that phosphorylation of activated PKC mu caused its reduced interaction with the antibody sc-639. Therefore, this antibody might present a well suited tool for the detection of activated PKC mu in vivo. Moreover, our results clearly show that some antibodies, such as sc-639, might be able to selectively detect non-phosphorylated or phosphorylated forms of a protein, and that such properties of an antibody have to be studied carefully before the latter can be used for reliable quantitative determination of this protein. We consider this information important to avoid misinterpretation of data concerning the immunological quantification of proteins such as PKC mu.

Differential effects of suramin on protein kinase C isoenzymes. A novel tool for discriminating protein kinase C activities

Suramin, a hexasulfonated naphthylurea, is known to induce differentiation and inhibit proliferation, angiogenesis, and development of tumors. It has also been shown to suppress the activity of the protein kinase C (PKC) isoenzymes α, β, and γ. Here we report on a differential effect of suramin on PKCμ and various PKC isoforms representing the cPKC, nPKC, and aPKC group of the PKC family. In the absence of any cofactors suramin activates all PKC isoforms in the order of aPKCζ≫PKCμ>cPKC, nPKCδ. As judged by the V max/K M ratios (0.5 for PKCμ and 2.2 for PKCζ) the substrate syntide 2 is phosphorylated by suramin‐activated PKCζ around four times more effectively than by suramin‐activated PKCμ. Suramin‐activated PKCμ behaves like that activated by phosphatidylserine and the phorbol ester TPA regarding autophosphorylation and differential inhibition by the PKC inhibitors Gö 6976 and Gö 6983. In the presence of activating cofactors, such as phosphatidylserine and TPA or cholesterol sulfate (for PKCζ), the activity of the aPKCζ is further stimulated, PKCμ is not significantly affected, and the cPKCs and the nPKCδ are strongly inhibited by suramin. The differential action of suramin on PKC isoenzymes might play a role in some of its biological effects, as for instance inhibition of proliferation and tumor development. Moreover, due to this property suramin will possibly be a valuable tool for discriminating the activities of PKC isoenzymes in vitro and in vivo.