Induction of beige‐like adipocyte markers and functions in 3T3‐L1 cells by Clk1 and PKCβII inhibitory molecules

Excessive dietary intake of fat results in its storage in white adipose tissue (WAT). Energy expenditure through lipid oxidation occurs in brown adipose tissue (BAT). Certain WAT depots can undergo a change termed beiging where markers that BAT express are induced. Little is known about signalling pathways inducing beiging. Here, inhibition of a signalling pathway regulating alternative pre‐mRNA splicing is involved in adipocyte beiging. Clk1/2/4 kinases regulate splicing by phosphorylating factors that process pre‐mRNA. Clk1 inhibition by TG003 results in beige‐like adipocytes highly expressing PGC1α and UCP1. SiRNA for Clk1, 2 and 4, demonstrated that Clk1 depletion increased UCP1 and PGC1α expression, whereas Clk2/4 siRNA did not. TG003‐treated adipocytes contained fewer lipid droplets, are smaller, and contain more mitochondria, resulting in proton leak increases. Additionally, inhibition of PKCβII activity, a splice variant regulated by Clk1, increased beiging. PGC1α is a substrate for both Clk1 and PKCβII kinases, and we surmised that inhibition of PGC1α phosphorylation resulted in beiging of adipocytes. We show that TG003 binds Clk1 more than Clk2/4 through direct binding, and PGC1α binds to Clk1 at a site close to TG003. Furthermore, we show that TG003 is highly specific for Clk1 across hundreds of kinases in our activity screen. Hence, Clk1 inhibition becomes a target for induction of beige adipocytes.     

Potent and selective small molecule inhibitors of specific isoforms of Cdc2-like kinases (Clk) and dual specificity tyrosine-phosphorylation-regulated kinases (Dyrk)

Continued examination of substituted 6-arylquinazolin-4-amines as Clk4 inhibitors resulted in selective inhibitors of Clk1, Clk4, Dyrk1A and Dyrk1B. Several of the most potent inhibitors were validated as being highly selective within a comprehensive kinome scan.     

Small-molecule pyrimidine inhibitors of the cdc2-like (Clk) and dual specificity tyrosine phosphorylation-regulated (Dyrk) kinases: Development of chemical probe ML315

Substituted pyrimidine inhibitors of the Clk and Dyrk kinases have been developed, exploring structure–activity relationships around four different chemotypes. The most potent compounds have low-nanomolar inhibitory activity against Clk1, Clk2, Clk4, Dyrk1A and Dyrk1B. Kinome scans with 442 kinases using agents representing three of the chemotypes show these inhibitors to be highly selective for the Clk and Dyrk families. Further off-target pharmacological evaluation with ML315, the most selective agent, supports this conclusion.     

Novel SR-rich-related protein clasp specifically interacts with inactivated Clk4 and induces the exon EB inclusion of Clk

We identified a novel serine/arginine (SR)-rich-related protein as a binding partner of Clk4 mutant lacking kinase activity (Clk4 K189R) in the two-hybrid screen and designated it Clasp (Clk4-associating SR-related protein). Northern blot analysis revealed that Clasp mRNA was highly expressed in brain, and in situ hybridization of a mouse brain sagittal section hybridized with antisense probes revealed that both Clasp and Clk4 mRNAs were expressed in the hippocampus, the cerebellum, and the olfactory bulb. Two forms of Clasp were produced by a frameshift following alternative splicing. The staining of an HA-tagged short form of Clasp (ClaspS) showed a nucleoplasmic pattern, while the long form of Clasp (ClaspL) was localized as nuclear dots. In vitro protein interaction assay demonstrated that Clk4 K189R was bound to Clasp while wild Clk4 was not. Overexpression of ClaspL promoted accumulation of Clk4 K189R in the nuclear dots and the exon EB inclusion from CR-1 and CR-2 pre-mRNA of Clk1. These data suggest that Clasp is a binding partner of Clk4 and may be involved in the regulation of the activity of Clk kinase family.     

Purification and properties of l(+)-lactate dehydrogenase from potato tubers

1. A purification of l(+)-lactate dehydrogenase is described. 2. The final preparation is active with NADH and NADPH and with a number of keto acids, but evidence is presented to support the view that a single enzyme is involved. 3. NAD(+) showed product inhibition, but at slightly acid pH values there was evidence of co-operative binding. 4. At acid pH values ATP was a potent inhibitor and appears to be an allosteric effector. At neutral or alkaline pH values ATP behaved as a weak competitive inhibitor. 5. The physiological significance of inhibition by ATP is discussed.     

Legionella translocates an E3 ubiquitin ligase that has multiple U-boxes with distinct functions

Legionella pneumophila has a Dot/Icm type IV secretion system used to translocate a number of 'effector proteins' which subvert host cell functions. In this study, we identified 19 novel Dot/Icm substrate proteins using a systematic screening technique. A blast analysis revealed that one of the substrates, which we named LubX ( L egionella U - b o x protein), contains two domains that have a remarkable similarity to the U-box, a domain found in eukaryotic E3 ubiquitin ligases. The expression of LubX is induced upon infection, and most of the LubX produced was translocated into the host cells. LubX has ubiquitin ligase activity in conjunction with UbcH5a or UbcH5c E2 enzymes and mediates polyubiquitination of host Clk1 (Cdc2-like kinase 1). We demonstrate that one of the U-boxes (U-box 1) is critical to the ubiquitin ligation, and the other U-box (U-box 2) mediates interaction with Clk1. Thus, the two U-boxes of LubX have distinct functions, and U-box 2 plays a non-canonical role in substrate binding. Although we demonstrate that inhibition of Clk kinase results in a marked reduction of Legionella growth within mouse macrophages, the consequence of Clk1 ubiquitination is still being elucidated. Together, these data suggest that Clk1 is the target host molecule which Legionella modulates during infection.     

SU9516: biochemical analysis of cdk inhibition and crystal structure in complex with cdk2

SU9516 is a 3-substituted indolinone compound with demonstrated potent and selective inhibition toward cyclin dependent kinases (cdks). Here, we describe the kinetic characterization of this inhibition with respect to cdk2, 1, and 4, along with the crystal structure in complex with cdk2. The molecule is competitive with respect to ATP for cdk2/cyclin A, with a K(i) value of 0.031 microM. Similarly, SU9516 inhibits cdk2/cyclin E and cdk1/cyclin B1 in an ATP-competitive manner, although at a 2- to 8-fold reduced potency. In contrast, the compound exhibited non-competitive inhibition with respect to ATP toward cdk4/cyclin D1, with a 45-fold reduced potency. The X-ray crystal structure of SU9516 bound to cdk2 revealed interactions between the molecule and Leu83 and Glu81 of the kinase. This study should aid in the development of more potent and selective cdk inhibitors for potential therapeutic agents.     

Cdc2-like kinase 2 (Clk2) promotes early neural development in Xenopus embryos

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways.     

L-Lactate dehydrogenase from leaves of higher plants. Kinetics and regulation of the enzyme from lettuce (Lactuca sativa L).

1. L-Lactate dehydrogenase from lettuce (Lactuca sativa) leaves was purified to electrophoretic homogeneity by affinity chromatography. 2. In addition to its NAD(H)-dependent activity with L-lactate and pyruvate, the enzyme also catalyses the reduction of hydroxypyruvate and glyoxylate. The latter activities are not due to a contamination of the enzyme preparations with hydroxypyruvate reductase. 3. The enzyme shows allosteric properties that are markedly by the pH. 4. ATP is a potent inhibitor of the enzyme. The kinetic data suggest that the inhibition by ATP is competitive with respect to NADH at pH 7.0 and 6.2. The existence of regulatory binding sites for ATP and NADH is discussed. 5. Bivalent metal cations and fructose 6-phosphate relieve the ATP inhibition of the enzyme. 6. A function of leaf L-lactate dehydrogenase is proposed as a component of the systems regulating the cellular pH and/or controlling the concentration of reducing equivalents in the cytoplasm of leaf cells.     

Cloning of human PRP4 reveals interaction with Clk1.

Prp4 is a protein kinase of Schizosaccharomyces pombe identified through its role in pre-mRNA splicing, and belongs to a kinase family including mammalian serine/arginine-rich protein-specific kinases and Clks, whose substrates are serine/arginine-rich proteins. We cloned human PRP4 (hPRP4) full-length cDNA and the antiserum raised against a partial peptide of hPRP4 recognized 170-kDa polypeptide in HeLa S3 cell extracts. Northern blot analysis revealed that hPRP4 mRNA was ubiquitously expressed in multiple tissues. The extended NH(2)-terminal region of hPRP4 contains an arginine/serine-rich domain and putative nuclear localization signals. hPRP4 phosphorylated and interacted with SF2/ASF, one of the essential splicing factors. Indirect immunofluorescence analysis revealed that endogenous hPRP4 was distributed in a nuclear speckled pattern and colocalized with SF2/ASF in HeLa S3 cells. Furthermore, hPRP4 interacted directly with Clk1 on its COOH terminus, and the arginine/serine-rich domain of hPRP4 was phosphorylated by Clk1 in vitro. Overexpression of Clk1 caused redistribution of hPRP4, from the speckled to the diffuse pattern in nucleoplasm, whereas inactive mutant of Clk1 caused no change of hPRP4 localization. These findings suggest that the NH(2)-terminal region of hPRP4 may play regulatory roles under an unidentified signal transduction pathway through Clk1.     

In vivo regulation of alternative pre-mRNA splicing by the Clk1 protein kinase.

Controlled expression of cellular and viral genes through alternative precursor messenger RNA (pre-mRNA) splicing requires serine/arginine-rich (SR) proteins. The Clk1 kinase, which phosphorylates SR proteins, is regulated through alternative splicing of the Clk1 pre-mRNA, yielding mRNAs encoding catalytically active and truncated inactive polypeptides (Clk1 and Clk1T, respectively). We present evidence that Clk1 and Clk1T proteins regulate the splicing of Clk1 and adenovirus pre-mRNAs in vivo. The peptide domain encoded by the alternatively spliced exon of Clk1 is essential for the regulatory activity of the Clk1 kinase. This is the first direct demonstration of an in vivo link between alternative splicing and protein kinase activity.     

Kinetic characterization of phosphofructokinase isolated from rat kidney cortex.

1. Phosphofructokinase from rat kidney cortex has been purified by affinity chromatography to a final specific activity of 15 units per mg of protein, measured at 25 degrees C and pH 8. 2. This lower spec. act., compared with that of the enzyme from other sources, shows the enzyme in proximal tubules to be less active, which would account for the main gluconeogenic role of these nephron sections. 3. The binding of fructose-6-phosphate to the enzyme is co-operative. ATP increases the Hill coefficient and produces a marked allosteric inhibition on the activity. 4. Fructose-2,6-bis-phosphate is a potent activator of the enzyme from this source. It reduces the Hill coefficient of the enzyme and the inhibition constant of ATP. A marked difference between this and the liver enzyme is that the activation is not co-operative.     

The Clk2 and Clk3 Dual-Specificity Protein Kinases Regulate the Intranuclear Distribution of SR Proteins and Influence Pre-mRNA Splicing

The three members of the Clk family of kinases (Clk1, 2, and 3) have been shown to undergo conserved alternative splicing to generate catalytically active (Clk) and inactive (Clk^T) isoforms. The prototype, murine Clk1 (mClk1), is a nuclear dual-specificity kinase that can interact with, and cause the nuclear redistribution of, SR proteins. In this study, we demonstrate that the human Clk2 and Clk3 (hClk2 and 3) are also found within the nucleus and display dual-specificity kinase activity. The truncated isoforms, hClk2^Tand hClk3^T, colocalize with SR proteins in nuclear speckles. We also show catalytically active hClk2 and hClk3 cause the redistribution of SR proteins and can regulate the alternative splicing of a model precursor mRNA substratein vivo.