Michael Gschwendt

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.

Inhibition of the calcium- and phospholipid-dependent protein kinase activity from mouse brain cytosol by quercetin

The flavonoid quercetin is a potent inhibitor of calcium- and phospholipid-dependent protein kinase (Ca, PL-PK) activity from mouse brain. Half-maximal inhibition of the kinase occurs at about 10 microM. If the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is used instead of calcium as a stimulating factor of the kinase enzyme activity is still inhibited by quercetin. The kinase inhibitor, however, does not interfere with the binding of TPA to its receptor.

Protein kinase C activation by phorbol esters: do cysteine-rich regions and pseudosubstrate motifs play a role?

A model for the binding of two activators of protein kinase C (PKC), the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and diacylglycerol, to the enzyme is proposed. It is suggested that each activator is hydrogen-bonded to sulfhydryl groups of cysteine residues and to the carbonyl of an asparagine within the cysteine-rich regions of PKC. This might induce a conformational change that would disrupt the association of the inhibitory pseudosubstrate sequence with the active center of PKC.

RIP4 (DIK/PKK), a novel member of the RIP kinase family, activates NF-κB and is processed during apoptosis

RIP1 and its homologs, RIP2 and RIP3, form part of a family of Ser/Thr kinases that regulate signal transduction processes leading to NF‐κB activation. Here, we identify RIP4 (DIK/PKK) as a novel member of the RIP kinase family. RIP4 contains an N‐terminal RIP‐like kinase domain and a C‐terminal region characterized by the presence of 11 ankyrin repeats. Overexpression of RIP4 leads to activation of NF‐κB and JNK. Kinase inactive RIP4 or a truncated version containing the ankyrin repeats have a dominant negative (DN) effect on NF‐κB induction by multiple stimuli. RIP4 binds to several members of the TRAF protein family, and DN versions of TRAF1, TRAF3 and TRAF6 inhibit RIP4‐induced NF‐κB activation. Moreover, RIP4 is cleaved after Asp340 and Asp378 during Fas‐induced apoptosis. These data suggest that RIP4 is involved in NF‐κB and JNK signaling and that caspase‐dependent processing of RIP4 may negatively regulate NF‐κB‐dependent pro‐survival or pro‐inflammatory signals.

Tyrosine phosphorylation and stimulation of protein kinase Cδ from porcine spleen by src in vitro

Native protein kinase Cδ from porcine spleen is phosphorylated in vitro by the tyrosine kinase src and to a much smaller extent by fyn. The tyrosine phosphorylation of PKCδ is restricted to the activated state of the enzyme, i.e. it occurs only in the presence of an activator, such as TPA or bryostatin. Upon phosphorylation at tyrosine, the apparent molecular weight of PKCδ increases by 6 kDa. Phosphorylation by src induces a stimulation of PKCδ activity apparently exhibiting some substrate selectivity. Other PKC isoenzymes, such as cPKC (α,β,γ), are not phosphorylated by src or only to a very small extent. This phosphorylation is not dependent on TPA and does not cause an increase in activity and molecular weight of the enzyme.

Conventional PKC-alpha, novel PKC-epsilon and PKC-theta, but not atypical PKC-lambda are MARCKS kinases in intact NIH 3T3 fibroblasts.

Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in intact cells has been employed as an indicator for activation of protein kinase C (PKC). Specific PKC isoenzymes responsible for MARCKS phosphorylation under physiological conditions, however, remained to be identified. In our present study using stably transfected NIH 3T3 cell clones we demonstrate that expression of constitutively active mutants of either conventional cPKC-α or novel nPKC-ε increased phosphorylation of endogenous MARCKS in the absence of phorbol 12,13-dibutyrate in intact mouse fibroblasts, implicating that each of these PKC isoforms itself is sufficient to induce enhanced MARCKS phosphorylation. Similarly, ectopic expression of a constitutively active mutant of PKC-η significantly increased MARCKS phosphorylation compared to vector controls, identifying PKC-η as a MARCKS kinase. The PKC-specific inhibitor GF 109203X (bisindolylmaleimide I) reduced MARCKS phosphorylation in intact cells at a similar dose-response as enzymatic activity of recombinant isoenzymes cPKC-α, nPKC-ε, and nPKC-η in vitro Consistently, phorbol 12,13-dibutyrate-dependent MARCKS phosphorylation was significantly reduced in cell lines expressing dominant negative mutants of either PKC-α K368R or (dominant negative) PKC-ε K436R. The fact, that the constitutively active PKC-λ A119E mutant did not alter the MARCKS phosphorylation underscores the assumption that atypical PKC isoforms are not involved in this process. We conclude that under physiological conditions, conventional cPKC-α and novel nPKC-ε, but not atypical aPKC-λ are responsible for MARCKS phosphorylation in intact NIH 3T3 fibroblasts.

Phosphorylation of Protein Kinase Cδ (PKCδ) at Threonine 505 Is Not a Prerequisite for Enzymatic Activity EXPRESSION OF RAT PKCδ AND AN ALANINE 505 MUTANT IN BACTERIA IN A FUNCTIONAL FORM

A structural feature shared by many protein kinases is the requirement for phosphorylation of threonine or tyrosine in the so-called activation loop for full enzyme activity. Previous studies by several groups have indicated that the isotypes α, βI, and βII of protein kinase C (PKC) are synthesized as inactive precursors and require phosphorylation by a putative “PKC kinase” for permissive activation. Expression of PKCα in bacteria resulted in a nonfunctional enzyme, apparently due to lack of this kinase. The phosphorylation sites for the PKC kinase in the activation loop of PKCα and PKCβII could be identified as Thr497 and Thr500, respectively. We report here that PKCδ, contrary to PKCα, can be expressed in bacteria in a functional form. The activity of the recombinant enzyme regarding substrate phosphorylation, autophosphorylation, and dependence on activation by 12-O-tetradecanoylphorbol-13-acetate as well as the Km values for two substrates are comparable to those of recombinant PKCδ expressed in baculovirus-infected insect cells. By site-directed mutagenesis we were able to show that Thr505, corresponding to Thr497 and Thr500 of PKCα and PKCβII, respectively, is not essential for obtaining a catalytically competent conformation of PKCδ. The mutant Ala505 can be activated and does not differ from the wild type regarding activity and several other features. Ser504 can not take over the role of Thr505 and is not prerequisite for the kinase to become activated, as proven by the unaffected enzyme activity of respective mutants (Ala504 and Ala504/Ala505). These results indicate that phosphorylation of Thr505 is not required for the formation of functional PKCδ and that at least this PKC isoenzyme differs from the isotypes α, βI, and βII regarding the permissive activation by a PKC kinase.

Calcium and phospholipid-dependent protein kinase activity in mouse epidermis cytosol. Stimulation by complete and incomplete tumor promoters and inhibition by various compounds.

Calcium- and phospholipid-dependent protein kinase (Ca, PL-PK) activity is detectable in mouse epidermis cytosol. It can be stimulated in vitro by complete and incomplete tumor promoters (12-0-tetradecanoylphorbol-13-acetate (TPA) and 12-0-retinoylphorbol-13-acetate (RPA], respectively. Effective inhibition of the enzyme activity is achieved with quercetin and phloretin, whereas the lipoxygenase and cyclooxygenase inhibitors nordihydroguaiaretic acid (NDGA) and esculetin show just weak or no inhibition. Quercetin inhibits the lipoxygenase and cyclooxygenase equally well as the Ca, PL-PK, whereas the strong Ca, PL-PK inhibitor phloretin is absolutely ineffective in inhibiting the lipoxygenase/cyclooxygenase. The application of these inhibitors in differentiating tumor promoter induced effects in vivo is proposed.

The mouse ear edema: A quantitatively evaluable assay for tumor promoting compounds and for inhibitors of tumor promotion

The induction of edema in the mouse ear has been established as a reliable in vivo assay for tumor promoting compounds and for inhibitors of tumor promotion. Apparently all skin tumor promoting compounds induce an edema, and all inhibitors of a tumor promoter-induced edema are most likely inhibitors of skin tumor promotion. No exception to this rule has been found as yet. Besides the application of this assay in the screening of compounds, the assay allows comparison of various compounds (e.g. complete and incomplete tumor promoters) with respect to their kinetics and dosage response in the induction of edema.

Protein kinase Cζ and n in murine epidermis TPA induces down‐regulation of PKCn but not PKCζ

Murine epidermis contains PKCζ and n as evidenced by the application or specific antisera. PKCζ predominates in the cytosol and PKCn in the particulate fraction. PKCζ is shown to be present also in other murine tissues, with large amounts found in lung. Whereas epidermal PKCn is completely down‐regulated by treatment or mouse skin with TPA or bryostatin 1 for 18 h. PKCζ is neither translocated by treatment with TPA for 20 min, nor down‐regulated by treatment with TPA or bryostatin 1 for 18 h. PKCζ is activated by phosphatidyl serine alone and does neither respond to Ca2+ nor to TPA. It is inhibited by staurosporine with an IC50 of 16 nM, which is within the same range or other PKC isoenzymes. The sensitivity of PKCζ towards the staurosporine derivative K252a is similar to that or PKCα,β,γ but much higher than that of PKCδ and ε.