A phase I, dose-escalation study of the Eg5-inhibitor EMD 534085 in patients with advanced solid tumors or lymphoma.

BACKGROUND: The kinesin spindle protein Eg5 is involved in mitosis, and its inhibition promotes mitotic arrest. EMD 534085, a potent, reversible Eg5 inhibitor, demonstrated significant preclinical antitumor activity. METHODS: This first-in-man, single-center, open-label, phase I dose-escalation study (3 + 3 design) investigated EMD 534085 safety, pharmacokinetics and antitumor activity in refractory solid tumors, Hodgkin's lymphoma, or non-Hodgkin's lymphoma. EMD 534085 (starting dose 2 mg/m²/day) was administered intravenously every 3 weeks. Doses were escalated in 100% steps in successive cohorts of 3 patients until grade 2 toxicity occurred, followed by 50% until the first dose-limiting toxicity (DLT) arose. If <2 of 6 patients experienced a DLT, doses were further increased by 25%. Dose-escalation was stopped if a DLT occurred in ≥2 of 6 patients. RESULTS: Forty-four patients received EMD 534085. Median treatment duration was 43 days (range, 21-337). Thirty-eight patients (86%) received ≥2 cycles. DLTs were grade 4 neutropenia (1 patient each at 108 and 135 mg/m²/day), and grade 3 acute coronary syndrome with troponin I elevation (1 patient at 135 mg/m²/day). The maximum tolerated dose (MTD) was 108 mg/m²/day. The most common treatment-related adverse events were asthenia (50%) and neutropenia (32%). EMD 534085 appeared to have linear pharmacokinetics. Increase in phospho-histone H3 positive cells in paired pre- and on-treatment biopsies showed evidence of target modulation. No complete or partial responses were observed. Best response was stable disease in 23 patients (52%). CONCLUSIONS: EMD 534085 appeared to be well tolerated; MTD was 108 mg/m²/day. Preliminary antitumor results suggested limited activity in monotherapy.

p21-activated kinase (PAK1) is phosphorylated and activated by 3-phosphoinositide-dependent kinase-1 (PDK1).

In this study, we show that phosphorylated 3-phosphoinositide-dependent kinase 1 (PDK1) phosphorylates p21-activated kinase 1 (PAK1) in the presence of sphingosine. We identify threonine 423, a conserved threonine in the activation loop of kinase subdomain VIII, as the PDK1 phosphorylation site on PAK1. Threonine 423 is a previously identified PAK1 autophosphorylation site that lies within a PAK consensus phosphorylation sequence. After pretreatment with phosphatases, autophosphorylation of PAK1 occurred at all major sites except threonine 423. A phosphothreonine 423-specific antibody detected phosphorylation of recombinant, catalytically inactive PAK1 after incubation with wild-type PAK1, indicating phosphorylation of threonine 423 occurs by an intermolecular mechanism. The biological significance of PDK1 phosphorylation of PAK1 at threonine 423 in vitro is supported by the observation that these two proteins interact in vivo and that PDK1-phosphorylated PAK1 has an increased activity toward substrate. An increase of phosphorylation of catalytically inactive PAK1 was observed in COS-7 cells expressing wild-type, but not catalytically inactive, PDK1 upon elevation of intracellular sphingosine levels. PDK1 phosphorylation of PAK1 was not blocked by pretreatment with wortmannin or when PDK1 was mutated to prevent phosphatidylinositol binding, indicating this process is independent of phosphatidylinositol 3-kinase activity. The data presented here provide evidence for a novel mechanism for PAK1 regulation and activation.

Sphingosine is a novel activator of 3-phosphoinositide-dependent kinase 1.

3-Phosphoinositide-dependent kinase 1 (PDK1) has previously been shown to phosphorylate the activation loop of several AGC kinase family members. In this study, we show that p21-activated kinase 1, the activity of which is regulated by the GTP-bound form of Cdc42 and Rac and by sphingosine, is phosphorylated by PDK1. Phosphorylation of p21-activated kinase 1 by PDK1 occurred only in the presence of sphingosine, which increased PDK1 autophosphorylation 25-fold. Sphingosine increased PDK1 autophosphorylation in a concentration-dependent manner and significantly increased phosphate incorporation into known PDK1 substrates. Studies on the lipid requirement for PDK1 activation found that both sphingosine isoforms and stearylamine also increased PDK1 autophosphorylation. However, C(10)-sphingosine, octylamine, and stearic acid were unable to increase PDK1 autophosphorylation, indicating that both a positive charge and a lipid tail containing at least a C(10)-carbon backbone were required for PDK1 activation. Three PDK1 autophosphorylation sites were identified after stimulation with sphingosine in a serine-rich region located between the kinase domain and the pleckstrin homology domain using two-dimensional phosphopeptide maps and matrix assisted laser desorption/ionization mass spectroscopy. Increased phosphorylation of endogenous Akt at threonine 308 was observed in COS-7 cells expressing wild type PDK1, but not catalytically inactive PDK1, when cellular sphingosine levels were elevated by treatment with sphingomyelinase. Sphingosine thus appears to be a true PDK1 activator.

Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity.

p21-activated kinases (Pak)/Ste20 kinases are regulated in vitro and in vivo by the small GTP-binding proteins Rac and Cdc42 and lipids, such as sphingosine, which stimulate autophosphorylation and phosphorylation of exogenous substrates. The mechanism of Pak activation by these agents remains unclear. We investigated Pak kinase activation in more detail to gain insight into the interplay between the GTPase/sphingosine binding, an intramolecular inhibitory interaction, and autophosphorylation. We present biochemical evidence that an autoinhibitory domain (ID) contained within amino acid residues 67-150 of Pak1 interacts with the carboxyl-terminal kinase domain and that this interaction is regulated in a GTPase-dependent fashion. Cdc42- and sphingosine-stimulated Pak1 activity can be inhibited in trans by recombinant ID peptide, indicating similarities in their mode of activation. However, Pak1, which was autophosphorylated in response to either GTPase or sphingosine, is highly active and is insensitive to inhibition by the ID peptide. We identified phospho-acceptor site threonine 423 in the kinase activation loop as a critical determinant for the sensitivity to autoinhibition and enzymatic activity. Phosphorylation studies suggested that the stimulatory effect of both GTPase and sphingosine results in exposure of the activation loop, making it accessible for intermolecular phosphorylation.

Regulated phosphorylation of the Gal4p inhibitor Gal80p of Kluyveromyces lactis revealed by mutational analysis.

The yeast Gal80 protein inhibits the transcription activation function of Gal4p by physically interacting with the activation domain (Gal4-AD). Gal80p interaction with Gal1p or Gal3p is required to relieve Gal4p inhibition in response to galactose. Gal80p orthologs of Saccharomyces cerevisiae and Kluyveromyces lactis, ScGal80p and KIGal80p, can also inhibit the heterologous Gal4p variants; however, heterologous Gal3p/Gal1p only regulate ScGal80p but not KIGal80p. To compare KIGal80p and ScGal80p, point mutations known to affect ScGal80p function were introduced at corresponding positions in KIGal80p, and Gal4p regulation in vivo and KIGal80p-binding to Gst-Gal1p and Gst-Gal4-AD in vitro were analysed. The in vitro binding properties of the KIGal80p mutants were similar to those of ScGal80p, but two out of four mutants differed in Gal4p regulation. E. g. KIGAL80s-0(G302R) but not ScGAL80s-0 (G301R) alleviates Gal4p inhibition. Possibly, this difference is related to a role of phosphorylation in the regulation of Gal80p function in K. lactis. Wild-type and mutant forms of KIGal80p are shown to be subject to carbon source regulated phosphorylation whereas no evidence for ScGal80p phosphorylation exists. (Hyper-)phosphorylation of KIGal80p is strongly reduced in galactose-containing medium. This reduction requires KIGal1p but no interaction with KIGal4p. The inhibition deficient KIGal80s-0p (G302R) variant is under-phosphorylated. We thus propose that phosphorylation of Gal80p in Kluyveromyces lactis contributes to the regulation of Gal4p mediated transcription.

alphaPix stimulates p21-activated kinase activity through exchange factor-dependent and -independent mechanisms.

Activation of p21-activated kinases (Paks) is achieved through binding of the GTPases Rac or Cdc42 to a conserved domain in the N-terminal regulatory region of Pak. Additional signaling components are also likely to be important in regulating Pak activation. Recently, a family of Pak-interacting guanine nucleotide exchange factors (Pix) have been identified and which are good candidates for regulating Pak activity. Using an active, truncated form of alphaPix (amino acids 155-545), we observe stimulation of Pak1 kinase activity when alphaPix155-545 is co-expressed with Cdc42 and wild-type Pak1 in COS-1 cells. This activation does not occur when we co-express a Pak1 mutant unable to bind alphaPix. The activation of wild-type Pak1 by alphaPix155-545 also requires that alphaPix155-545 retain functional exchange factor activity. However, the Pak1(H83,86L) mutant that does not bind Rac or Cdc42 is activated in the absence of GTPase by alphaPix155-545 and by a mutant of alphaPix155-545 that no longer has exchange factor activity. Pak1 activity stimulated in vitro using GTPgammaS-loaded Cdc42 was also enhanced by recombinant alphaPix155-545 in a binding-dependent manner. These data suggest that Pak activity can be modulated by physical interaction with alphaPix and that this specific effect involves both exchange factor-dependent and -independent mechanisms.

p21-activated kinase (PAK) is required for Fas-induced JNK activation in Jurkat cells.

The process of apoptosis is a critical component of normal immune system development and homeostasis, and in many cells this involves signaling through the c-Jun amino terminal kinase (JNK) pathway. In Jurkat T cells, Fas-induced JNK activity is dependent upon activation of the caspase cascades known to be central components of the apoptotic program. We show in Jurkat cell lines expressing a dominant negative PAK construct that PAK signaling is necessary for JNK activation in response to Fas receptor cross-linking. Inhibition of JNK activation induced by Fas does not impair cell death as assessed by DNA fragmentation. However, expression of the catalytically active C terminus of PAK2, which is generated through caspase action during Fas-mediated apoptosis, induces Jurkat cell apoptosis. We conclude that PAK activity resulting from caspase-mediated cleavage is a necessary component of JNK activation induced by Fas receptor signaling and that PAK2 can contribute to the induction of cell death.

Activation of Gal4p by galactose-dependent interaction of galactokinase and Gal80p.

Yeast galactokinase (Gal1p) is an enzyme and a regulator of transcription. In addition to phosphorylating galactose, Gal1p activates Gal4p, the activator of GAL genes, but the mechanism of this regulation has been unclear. Here, biochemical and genetic evidence is presented to show that Gal1p activates Gal4p by direct interaction with the Gal4p inhibitor Gal80p. Interaction requires galactose, adenosine triphosphate, and the regulatory function of Gal1p. These data indicate that Gal1p-Gal80p complex formation results in the inactivation of Gal80p, thereby transmitting the galactose signal to Gal4p.

Nitric oxide destroys zinc-sulfur clusters inducing zinc release from metallothionein and inhibition of the zinc finger-type yeast transcription activator LAC9.

Nitric oxide, generated from S-nitrosocysteine or applied as gas mediates metal ion release from the Zn2+/Cd(2+)-complexing protein metallothionein via oxidation of SH-groups. Time-dependent S-nitrosylation and subsequent disulfide formation of metallothionein are demonstrated. Furthermore, nitric oxide inhibits DNA binding activity of the yeast transcription factor LAC9 containing a zinc finger like DNA binding domain. These results show that nitric oxide interacts with and destroys zinc-sulfur clusters in proteins.

Gal80 proteins of Kluyveromyces lactis and Saccharomyces cerevisiae are highly conserved but contribute differently to glucose repression of the galactose regulon.

We cloned the GAL80 gene encoding the negative regulator of the transcriptional activator Gal4 (Lac9) from the yeast Kluyveromyces lactis. The deduced amino acid sequence of K. lactis GAL80 revealed a strong structural conservation between K. lactis Gal80 and the homologous Saccharomyces cerevisiae protein, with an overall identity of 60% and two conserved blocks with over 80% identical residues. K. lactis gal80 disruption mutants show constitutive expression of the lactose/galactose metabolic genes, confirming that K. lactis Gal80 functions in essentially in the same way as does S. cerevisiae Gal80, blocking activation by the transcriptional activator Lac9 (K. lactis Gal4) in the absence of an inducing sugar. However, in contrast to S. cerevisiae, in which Gal4-dependent activation is strongly inhibited by glucose even in a gal80 mutant, glucose repressibility is almost completely lost in gal80 mutants of K. lactis. Indirect evidence suggests that this difference in phenotype is due to a higher activator concentration in K. lactis which is able to overcome glucose repression. Expression of the K. lactis GAL80 gene is controlled by Lac9. Two high-affinity binding sites in the GAL80 promoter mediate a 70-fold induction by galactose and hence negative autoregulation by Gal80. Gal80 in turn not only controls Lac9 activity but also has a moderate influence on its rate of synthesis. Thus, a feedback control mechanism exists between the positive and negative regulators. By mutating the Lac9 binding sites of the GAL80 promoter, we could show that induction of GAL80 is required to prevent activation of the lactose/galactose regulon in glycerol or glucose plus galactose, whereas the noninduced level of Gal80 is sufficient to completely block Lac9 function in glucose.