Mst1 and Mst2 kinases: regulations and diseases

The Hippo signaling pathway has emerged as a critical regulator for organ size control. The serine/threonine protein kinases Mst1 and Mst2, mammalian homologs of the Hippo kinase from Drosophila, play the central roles in the Hippo pathway controlling the cell proliferation, differentiation, and apoptosis during development. Mst1/2 can be activated by cellular stressors and the activation of Mst1/2 might enforce a feedback stimulation system to regulate oxidant levels through several mechanisms, in which regulation of cellular redox state might represent a tumor suppressor function of Mst1/2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in multiple organs, although considerable diversification in the pathway composition and regulation is observed in some of them. Generally, loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by the reduction or elimination of YAP. The Hippo pathway integrates with other signaling pathways e.g. Wnt and Notch pathways and coordinates with them to impact on the tumor pathogenesis and development. Furthermore, Mst1/2 kinases also act as an important regulator in immune cell activation, adhesion, migration, growth, and apoptosis. This review will focus on the recent updates on those aspects for the roles of Mst1/2 kinases.     

JNK phosphorylates Yes-associated protein (YAP) to regulate apoptosis

Yes-associated protein (YAP) regulates DNA damage and chemosensitivity, as well as functioning as a pro-growth, cell size regulator. For both of its roles, regulation by phosphorylation is crucial. We undertook an in vitro screen to identify novel YAP kinases to discover new signaling pathways to better understand YAP''s function. We identified JNK1 and JNK2 as robust YAP kinases, as well as mapped multiple sites of phosphorylation. Using inhibitors and siRNA, we showed that JNK specifically phosphorylates endogenous YAP in a number of cell types. We show that YAP protects keratinocytes from UV irradiation but promotes UV-induced apoptosis in a squamous cell carcinoma. We defined the mechanism for this dual role to be YAP''s ability to bind and stabilize the pro-proliferative ΔNp63α isoform in a JNK-dependent manner. Our report indicates that an evaluation of the expression of the different isoforms of p63 and p73 is crucial in determining YAP''s function.     

Phosphorylation target site specificity for AGC kinases DMPK E and Lats2

Serine/threonine kinases of the AGC group are important regulators of cell growth and motility. To examine the candidate substrate profile for two members of this group, DMPK E and Lats2, we performed in vitro kinase assays on peptide arrays. Substrate peptides for both kinases exhibited a predominance of basic residues surrounding the phosphorylation target site. 3D homology modeling of the kinase domains of DMPK E and Lats2 indicated that presence of two negative pockets in the peptide binding groove provides an explanation for the substrate preference. These findings will aid future research toward signaling functions of Lats2 and DMPK E within cells.     

Mitogen-activated protein kinases regulate COX-2 and mucosal recovery in ischemic-injured porcine ileum

Mitogen-activated protein kinase (MAPK) pathways transduce signals from a diverse array of extracellular stimuli. The three primary MAPK-signaling pathways are the extracellular regulated kinases (ERK1/2), p38 MAPK, and c-Jun NH(2)-terminal kinase (JNK). Previous research in our laboratory has shown that COX-2-elaborated prostanoids participate in recovery of mucosal barrier function in ischemic-injured porcine ileum. Because COX-2 expression is regulated in part by MAPKs, we postulated that MAPK pathways would play an integral role in recovery of injured mucosa. Porcine mucosa was subjected to 45 min of ischemia, after which tissues were mounted in Ussing chambers, and transepithelial electrical resistance (TER) was monitored as an index of recovery of barrier function. Treatment of tissues with the p38 MAPK inhibitor SB-203580 (0.1 mM) or the ERK1/2 inhibitor PD-98059 (0.1 mM) abolished recovery. Western blot analysis revealed that SB-203580 inhibited upregulation of COX-2 that was observed in untreated ischemic-injured mucosa, whereas PD-98059 had no effect on COX-2 expression. Inhibition of TER recovery by SB-203580 or PD-98059 was overcome by administration of exogenous prostaglandin E(2) (1 microM). The JNK inhibitor SP-600125 (0.1 mM) significantly increased TER and resulted in COX-2 upregulation. COX-2 expression appears to be positively and negatively regulated by the p38 MAPK and the JNK pathways, respectively. Alternatively, ERK1/2 appear to be involved in COX-2-independent reparative events that remain to be defined.     

Sequence analysis of cell cycle control (cdc2) protein kinases among protein serine/threonine kinases

Among protein serine/threonine kinases, the CDC2 proteins are both well characterized as protein serine/threonine kinases and are functionally involved in the control of cell division. Protein serine/threonine kinase sequences were analysed using Fourier transform of the coded sequences. Characteristic code/frequency pairs were extracted from a set of well defined protein serine/threonine kinases. The characteristic frequencies 0.179, 0.250 and 0.408 distinguished protein serine/threonine kinases from proteins which did not have the biological activity. Pertinent patterns in the sequence, responsible for the code/frequency pairs detection were searched and found to be correlated with the putative catalytic domain of the proteins. Protein serine/threonine kinases involved in cell division control, CDC2 protein kinases, were compared to the other protein serine/threonine kinases. Specific code/frequency pairs were extracted from the sequences and could be related to the function or regulation of the kinases in cell division. Two CDC2 related proteins CDC2(Mm) from mice and CDC2(Gg) from chicken were shown to fit well with the CDC2 proteins, whereas KIN28, PHO85 and PSKJ3, which share sequence homology but not functional activity with the CDC2 proteins, were clearly excluded from the CDC2 proteins by the characteristic code/frequency pairs. Pertinent patterns in the CDC2 proteins were analysed and mapped on the CDC2 related protein sequences. Four patterns were correlated with the code/frequency detection and therefore, could be associated to the regulation of the CDC2-related proteins.     

The WAG1 and WAG2 protein kinases negatively regulate root waving in Arabidopsis

The WAG1 and WAG2 genes of Arabidopsis thaliana encode protein-serine/threonine kinases that are closely related to PINOID. In order to determine what roles WAG1 and WAG2 play in seedling development, we used a reverse genetics approach to study the wag1, wag2 and wag1/wag2 mutant phenotypes for clues. Although the wag mutants do not contain detectable amounts of the corresponding mRNA, they are wild type in most respects. However, wag1/wag2 double mutants exhibit a pronounced wavy root phenotype when grown vertically on agar plates, a phenotype observed in wild-type plants only on plates inclined to angles less than 90 degrees. The wag1 and wag2 mutants also demonstrate enhanced root waving, but to a lesser extent. Moreover, the double mutant roots are more resistant to the effects of N-1-naphthylphthalamic acid on the inhibition of root curling, raising the possibility that transport of auxin is affected in the wag mutants. Promoter fusions to the gusA reporter gene demonstrate that the WAG promoters are most active in root tips, consistent with the observed phenotypes in the wag mutants.     

Tyrosine kinases regulate intracellular calcium during alpha(2)-adrenergic contraction in rat aorta

We have demonstrated enhanced contractile sensitivity to the alpha(2)-adrenoreceptor (alpha(2)-AR) agonist UK-14304 in arteries from rats made hypertensive with chronic nitric oxide synthase (NOS) inhibition (LHR) compared with arteries from normotensive rats (NR); additionally, this contraction requires Ca(2+) entry. We hypothesized that tyrosine kinases augment alpha(2)-AR contraction in LHR arteries by increasing Ca(2+). The tyrosine kinase inhibitor tyrphostin 23 significantly attenuated UK-14304 contraction of denuded thoracic aortic rings from NR and LHR. However, tyrphostin 23 did not alter UK-14304 contraction in ionomycin-permeabilized aorta, which indicates that tyrosine kinases regulate intracellular Ca(2+) concentration. The Src family inhibitor PP1 and the epidermal growth factor receptor kinase inhibitor AG-1478 did not alter alpha(2)-AR contraction, whereas the mitogen-activated protein kinase extracellular signal-regulated kinase kinase inhibitor PD-98059 attenuated the contraction. Contraction to CaCl(2) in ionomycin-permeabilized LHR rings was greater than in NR rings. UK-14304 augmented CaCl(2) contraction in ionomycin-permeabilized rings from both groups but to a greater extent in LHR aorta. Together, these data suggest that alpha(2)-AR stimulates contraction via two pathways. One, which is enhanced with NOS inhibition hypertension, activates Ca(2+) sensitivity and is independent of tyrosine kinases. The other is tyrosine kinase dependent and regulates intracellular Ca(2+) concentration.     

Structure and function of the protein tyrosine kinases

The protein tyrosine kinases (PTKs) are a large and structurally diverse family of enzymes. The conserved catalytic domain held in common by each member of this family is a self-contained 250–300 amino acid unit bearing sixteen highly conserved linear sequence elements, several of which have been shown to be important to the catalytic activity of this domain. The enzymic activity of the PTKs is clearly an evolutionarily successful theme, and at least 10 distinct morphotypes have been described. Many of these resemble cell surface receptors for growth factors, and for a small sub-set of these receptors a ligand has been discovered. The remainder are located intracellularly and presumably sense and respond to appropriate metabolic cues by exerting their physiologically powerful enzymic activity. A detailed examination of the structure/function relationships of the PTKs and their catalytic domains is particularly revealing in trying to establish the roles that these proteins play in signal transduction in eukaryotic cells.     

Association of mammalian sterile twenty kinases, Mst1 and Mst2, with hSalvador via C-terminal coiled-coil domains, leads to its stabilization and phosphorylation

Genetic screens in Drosophila have revealed that the serine/threonine kinase Hippo (Hpo) and the scaffold protein Salvador participate in a pathway that controls cell proliferation and apoptosis. Hpo most closely resembles the pro-apoptotic mammalian sterile20 kinases 1 and 2 (Mst1 and 2), and Salvador (Sav) has a human orthologue hSav (also called hWW45). Here we show that Mst and hSav heterodimerize in an interaction requiring the conserved C-terminal coiled-coil domains of both proteins. hSav was also able to homodimerize, but this did not require its coiled-coil domain. Coexpression of Mst and hSav led to phosphorylation of hSav and also increased its abundance. In vitro phosphorylation experiments indicate that the phosphorylation of Sav by Mst is direct. The stabilizing effect of Mst was much greater on N-terminally truncated hSav mutants, as long as they retained the ability to bind Mst. Mst mutants that lacked the C-terminal coiled-coil domain and were unable to bind to hSav, also failed to stabilize or phosphorylate hSav, whereas catalytically inactive Mst mutants that retained the ability to bind to hSav were still able to increase its abundance, although they were no longer able to phosphorylate hSav. Together these results show that hSav can bind to, and be phosphorylated by, Mst, and that the stabilizing effect of Mst on hSav requires its interaction with hSav but is probably not due to phosphorylation of hSav by Mst.     

Cyclin-dependent kinases regulate splice-specific targeting of dynamin-related protein 1 to microtubules

Fission and fusion reactions determine mitochondrial morphology and function. Dynamin-related protein 1 (Drp1) is a guanosine triphosphate–hydrolyzing mechanoenzyme important for mitochondrial fission and programmed cell death. Drp1 is subject to alternative splicing of three exons with previously unknown functional significance. Here, we report that splice variants including the third but excluding the second alternative exon (x01) localized to and copurified with microtubule bundles as dynamic polymers that resemble fission complexes on mitochondria. A major isoform in immune cells, Drp1-x01 required oligomeric assembly and Arg residues in alternative exon 3 for microtubule targeting. Drp1-x01 stabilized and bundled microtubules and attenuated staurosporine-induced mitochondrial fragmentation and apoptosis. Phosphorylation of a conserved Ser residue adjacent to the microtubule-binding exon released Drp1-x01 from microtubules and promoted mitochondrial fragmentation in a splice form–specific manner. Phosphorylation by Cdk1 contributed to dissociation of Drp1-x01 from mitotic microtubules, whereas Cdk5-mediated phosphorylation modulated Drp1-x01 targeting to interphase microtubules. Thus, alternative splicing generates a latent, cytoskeletal pool of Drp1 that is selectively mobilized by cyclin-dependent kinase signaling.     

Serum and glucocorticoid inducible kinases functionally regulate ClC-2 channels

ClC-2 participates in the regulation of neuronal excitability, chloride secretion, and cell volume. The ClC-2 sequence contains a consensus site (Ser82) for phosphorylation by the serum and glucocorticoid inducible kinase isoforms SGK1-3. Thus, the present study explored whether ClC-2 is regulated by those kinases. ClC-2 expression in Xenopus oocytes induced inwardly rectifying currents that increased upon coexpression of SGK1-3 and the related kinase PKB. The stimulatory effect was still present upon disruption of the SGK phosphorylation site. SGKs can phosphorylate the ubiquitin ligase Nedd4-2 and prevent Nedd4-2 from binding to its target. Therefore, the role of Nedd4-2 in ClC-2 modulation was investigated. ClC-2 activity decreased upon Nedd4-2 coexpression, an effect reversed by the kinases. According to chemiluminescence ClC-2 membrane abundance was enhanced by SGKs and diminished by Nedd4-2. These observations suggest that SGK1-3 and Nedd4-2 regulate ClC-2 at least in part by modulating ClC-2 abundance at the plasma membrane.     

Putative tumor suppressor Lats2 induces apoptosis through downregulation of Bcl-2 and Bcl-x(L)

Lats2, also known as Kpm, is the second mammalian member of the novel Lats tumor suppressor gene family. Recent studies have demonstrated that Lats2 negatively regulates the cell cycle by controlling G1/S and/or G2/M transition. To further understand the role of Lats2 in the control of human cancer development, we have expressed the protein in human lung cancer cells by transduction of a replication-deficient adenovirus expressing human Lats2 (Ad-Lats2). Using a variety of techniques, including Annexin V uptake, cleavage of PARP, and DNA laddering, we have demonstrated that the ectopic expression of human Lats2 induced apoptosis in two lung cancer cell lines, A549 and H1299. Caspases-3, 7, 8, and 9 were processed in the Ad-Lats2-transduced cells; however, it was active caspase-9, not caspase-8, that initiated the caspase cascade. Inhibitors specific to caspase-3 and 9 delayed the onset of Lats2-mediated apoptosis. Western blot analysis revealed that anti-apoptotic proteins, BCL-2 and BCL-x(L), but not the pro-apoptotic protein, BAX, were downregulated in Ad-Lats2-transduced human lung cancer cells. Overexpression of either Bcl-2 or Bcl-x(L) in these cells lead to the suppression of Lats2-mediated caspase cleavage and apoptosis. These results show that Lats2 induces apoptosis through downregulating anti-apoptotic proteins, BCL-2 and BCL-x(L), in human lung cancer cells.     

Abl kinases regulate autophagy by promoting the trafficking and function of lysosomal components

Autophagy is a lysosome-dependent degradative pathway that regulates the turnover of intracellular organelles, parasites, and long-lived proteins. Deregulation of autophagy results in a variety of pathological conditions, but little is known regarding the mechanisms that link normal cellular and pathological signals to the regulation of distinct stages in the autophagy pathway. Here we uncover a novel role for the Abl family kinases in the regulation of the late stages of autophagy. Inhibition, depletion, or knockout of the Abl family kinases, Abl and Arg, resulted in a dramatic reduction in the intracellular activities of the lysosomal glycosidases alpha-galactosidase, alpha-mannosidase and neuraminidase. Inhibition of Abl kinases also reduced the processing of the precursor forms of cathepsin D and cathepsin L to their mature, lysosomal forms, which coincided with the impaired turnover of long-lived cytosolic proteins and accumulation of autophagosomes. Furthermore, defective lysosomal degradation of long-lived proteins in the absence of Abl kinase signaling was accompanied by a perinuclear redistribution of lysosomes and increased glycosylation and stability of lysosome-associated membrane proteins, which are known to be substrates for lysosomal enzymes and play a role in regulating lysosome mobility. Our findings reveal a role for Abl kinases in the regulation of late-stage autophagy and have important implications for therapies that employ pharmacological inhibitors of the Abl kinases.     

Vanadium salts stimulate mitogen-activated protein (MAP) kinases and ribosomal S6 kinases

Effect of several vanadium salts, sodium orthovanadate, vanadyl sulfate and sodium metavanadate on protein tyrosine phosphorylation and serine/threonine kinases in chinese hamster ovary (CHO) cells overexpressing a normal human insulin receptor was examined. All the compounds stimulated protein tyrosine phosphorylation of two major proteins with molecular masses of 42 kDa (p42) and 44 kDa (p44). The phosphorylation of p42 and p44 was associated with an activation of mitogen activated protein (MAP) kinase as well as increased protein tyrosine phosphorylation of p42^mapk and p44^mapk. Vanadinm salts also activated the 90 kDa ribosomal s6 kinase (p90^rsk) and 70 kDa ribosomal s6 kinase (p70^s6k). Among the three vanadium salts tested, vanadyl sulfate appeared to be slightly more potent than others in stimulating MAP kinases and p70^s6k activity. It is suggested that vanadium-induced activation of MAP kinases and ribosomal s6 kinases may be one of the mechanisms by which insulin like effects of this trace element are mediated.Abbreviations eIF-4 eukaryotic protein synthesis initiation factor-4 - GRB-2 growth factor receptor bound protein-2 - GSK-3 Glycogen Synthase Kinase-3 - IRS-1 insulin receptor substrate-1 - ISPK insulin stimulated protein kinase - MAPK mitogen activated protein kinase, also known as - ERK extracellular signal regulated kinase - MAPKK mitogen activated protein kinase kinase, also known as-MEK, MAPK or ERK kinase - PHAS-1 phosphorylated heat and acid stable protein regulated by insulin - PI3K phosphatidyl inositol 3-kinase - PP1-G protein phosphatase-glycogen bound form - PTK protein tyrosine kinase - PTPase protein tyrosine phosphatase - rsk ribosomal s6 kinases - shc src homology domain containing protein - SOS son of sevenless     

S-phase kinase-associated protein 2 promotes cell growth and motility in osteosarcoma cells

Skp2 (S-phase kinase-associated protein 2) plays an oncogenic role in a variety of human cancers. However, the function of Skp2 in osteosarcoma (OS) is elusive. Therefore, in the current study, we explore whether Skp2 exerts its oncogenic function in OS. The cell growth, apoptosis, invasion and cell cycle were measured in OS cells after Skp2 overexpression. We found that overexpression of Skp2 enhanced cell growth, and inhibited cell apoptosis in OS cells. Moreover, we observed that upregulation of Skp2 accelerated cell cycle progression in OS cells. Furthermore, the ability of migration and invasion was enhanced in Skp2 overexpressing OS cells. Mechanically, our Western blotting data suggested that Skp2 decreased the expression of E-cadherin, Foxo1, p21, and p57, but increased MMP-9 in OS cells. In conclusion, our study demonstrated that Skp2 exhibited an oncogenic function in OS cells, suggesting that inhibition of Skp2 may be a novel approach for the treatment of OS.