Hyongjong Koh

Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production

The Akt/protein kinase B (PKB) serine/ threonine kinase is well known as an important mediator of many cell survival signaling pathways. Here, we demonstrate for the first time a major role of Akt/PKB in the cell invasion properties of the highly metastatic cell line HT1080. Using confocal microscopic analyses of live samples, we found Akt/PKB to be localized in the leading edge membrane area of migrating HT1080 cells. This localization was dependent on phosphoino‐sitide 3‐kinase and required the lipid binding ability of the phosphoinositide binding pleckstrin homology domain of Akt/PKB. We examined the possible function of Akt/PKB in HT1080 invasion. Surprisingly, Akt/ PKB potently promoted HT1080 invasion, by increasing cell motility and matrix metalloproteinase‐9 (MMP‐9) production, in a manner highly dependent on its kinase activity and membrane‐translocating ability. The increase in MMP‐9 production was mediated by activation of nuclear factor‐κB transcriptional activity by Akt/PKB. However, Akt/PKB did not affect the cell‐cell or cell‐matrix adhesion properties of HT1080. Our findings thus establish Akt/PKB as a major factor in the invasive abilities of cancer cells.

Energy-dependent regulation of cell structure by AMP-activated protein kinase

AMP-activated protein kinase (AMPK, also known as SNF1A) has been primarily studied as a metabolic regulator that is activated in response to energy deprivation. Although there is relatively ample information on the biochemical characteristics of AMPK, not enough data exist on the in vivo function of the kinase. Here, using the Drosophila model system, we generated the first animal model with no AMPK activity and discovered physiological functions of the kinase. Surprisingly, AMPK-null mutants were lethal with severe abnormalities in cell polarity and mitosis, similar to those of lkb1-null mutants. Constitutive activation of AMPK restored many of the phenotypes of lkb1-null mutants, suggesting that AMPK mediates the polarity- and mitosis-controlling functions of the LKB1 serine/threonine kinase. Interestingly, the regulatory site of non-muscle myosin regulatory light chain (MRLC; also known as MLC2) was directly phosphorylated by AMPK. Moreover, the phosphomimetic mutant of MRLC rescued the AMPK-null defects in cell polarity and mitosis, suggesting MRLC is a critical downstream target of AMPK. Furthermore, the activation of AMPK by energy deprivation was sufficient to cause dramatic changes in cell shape, inducing complete polarization and brush border formation in the human LS174T cell line, through the phosphorylation of MRLC. Taken together, our results demonstrate that AMPK has highly conserved roles across metazoan species not only in the control of metabolism, but also in the regulation of cellular structures.

Drosophila phosphoinositide-dependent kinase-1 regulates apoptosis and growth via the phosphoinositide 3-kinase-dependent signaling pathway

Phosphoinositide-dependent kinase-1 (PDK-1) is a central mediator of the cell signaling between phosphoinositide 3-kinase (PI3K) and various intracellular serine/threonine kinases including Akt/protein kinase B (PKB), p70 S6 kinases, and protein kinase C. Recent studies with cell transfection experiments have implied that PDK-1 may be involved in various cell functions including cell growth and apoptosis. However, despite its pivotal role in cellular signalings, the in vivo functions of PDK-1 in a multicellular system have rarely been investigated. Here, we have isolated Drosophila PDK-1 (dPDK-1) mutants and characterized the in vivo roles of the kinase. Drosophila deficient in the dPDK-1 gene exhibited lethality and an apoptotic phenotype in the embryonic stage. Conversely, overexpression of dPDK-1 increased cell and organ size in a Drosophila PI3K-dependent manner. dPDK-1 not only could activate Drosophila Akt/PKB (Dakt1), but also substitute the in vivo functions of its mammalian ortholog to activate Akt/PKB. This functional interaction between dPDK-1 and Dakt1 was further confirmed through genetic analyses in Drosophila. On the other hand, cAMP-dependent protein kinase, which has been proposed as a possible target of dPDK-1, did not interact with dPDK-1. In conclusion, our findings provide direct evidence that dPDK-1 regulates cell growth and apoptosis during Drosophila development via the PI3K-dependent signaling pathway and demonstrate our Drosophila system to be a powerful tool for elucidating the in vivo functions and targets of PDK-1.

Cloning and characterization of a nuclear S6 kinase, S6 kinase-related kinase (SRK); A novel nuclear target of Akt

Akt is stimulated by several growth factors, and mediates their cell survival signals. Recent studies have shown that Akt may play an intermediate role between phosphatidylinositol 3-kinase (PI3K) and p70 S6 kinase (p70S6K). Here we show that a novel nuclear p70S6K-related kinase (SRK) exists and that its in vivo function is also augmented by over-expression of Akt. Conceptual translation of the SRK cDNA revealed that the catalytic domain of SRK was highly homologous to that of p70S6K, and that the treatment of wortmannin or rapamycin strongly inhibited the phosphorylation and the activation of SRK, as in p70S6K. However, the N- and C-terminal domains of SRK were quite different from those of p70S6K. In immunolocalization analyses, we demonstrated a constitutive nuclear localization of SRK and the presence of a nuclear localization signal in its C-terminus. In vitro S6 phosphotransferase activities of SRK were stimulated with a slower kinetics by a variety of agonists to p70S6K. Interestingly, over-expression of the proto-oncogene Akt resulted in EGF-independent activation of SRK, while over-expression of kinase-dead Akt actually had an inhibitory effect. This relationship between Akt and SRK suggests that SRK may be a novel target of Akt and perhaps an important downstream component in the nuclear function of Akt.

Inhibition of Akt and Its Anti-apoptotic Activities by Tumor Necrosis Factor-induced Protein Kinase C-related Kinase 2 (PRK2) Cleavage

Akt is stimulated by several growth factors and has a major anti-apoptotic role in the cell. Therefore, we hypothesized that a pathway leading to the inhibition of Akt might be utilized in the process of apoptosis. Accordingly, we used a yeast two-hybrid screening assay to identify the proteins that interact with and possibly inhibit Akt. We found that the C-terminal region of protein kinase C-related kinase 2 (PRK2), containing amino acids 862 to 908, specifically binds to Akt in yeast and mammalian cells. During early stages of apoptosis, the C-terminal region of PRK2 is cleaved from the inhibitory N-terminal region and can bind Akt. The protein-protein interaction between Akt and the PRK2 C-terminal region specifically down-modulates the protein kinase activities of Akt by inhibiting phosphorylation at threonine 308 and serine 473 of Akt. This inhibition of Akt leads to the inhibition of the downstream signaling of Aktin vivo. The PRK2 C-terminal fragment strongly inhibits the Akt-mediated phosphorylation of BAD, a pro-apoptotic Bcl-2 family protein, and blocks the anti-apoptotic activities of Aktin vivo. These results provide direct evidence that the products of protein cleavage during apoptosis inhibit pro-survival signalings, leading to the amplification of pro-apoptotic signalings in the cell.

Inhibition of ERK-MAP kinase signaling by RSK during Drosophila development.

Although p90 ribosomal S6 kinase (RSK) is known as an important downstream effector of the ribosomal protein S6 kinase/extracellular signal‐regulated kinase (Ras/ERK) pathway, its endogenous role, and precise molecular function remain unclear. Using gain‐of‐function and null mutants of RSK, its physiological role was successfully characterized in Drosophila. Surprisingly, RSK‐null mutants were viable, but exhibited developmental abnormalities related to an enhanced ERK‐dependent cellular differentiation such as ectopic photoreceptor‐ and vein‐cell formation. Conversely, overexpression of RSK dramatically suppressed the ERK‐dependent differentiation, which was further augmented by mutations in the Ras/ERK pathway. Consistent with these physiological phenotypes, RSK negatively regulated ERK‐mediated developmental processes and gene expressions by blocking the nuclear localization of ERK in a kinase activity‐independent manner. In addition, we further demonstrated that the RSK‐dependent inhibition of ERK nuclear migration is mediated by the physical association between ERK and RSK. Collectively, our study reveals a novel regulatory mechanism of the Ras/ERK pathway by RSK, which negatively regulates ERK activity by acting as a cytoplasmic anchor in Drosophila.

JNK pathway mediates apoptotic cell death induced by tumor suppressor LKB1 in Drosophila

Although recent progresses have unveiled the diverse in vivo functions of LKB1, detailed molecular mechanisms governing these processes still remain enigmatic. Here, we showed that Drosophila LKB1 negatively regulates organ growth by caspase-dependent apoptosis, without affecting cell size and cell cycle progression. Through genetic screening for LKB1 modifiers, we discovered the JNK pathway as a novel component of LKB1 signaling; the JNK pathway was activated by LKB1 and mediated the LKB1-dependent apoptosis. Consistently, LKB1-null mutant was defective in embryonic apoptosis and displayed a drastic hyperplasia in the central nervous system; these phenotypes were fully rescued by ectopic JNK activation as well as wild-type LKB1 expression. Furthermore, inhibition of LKB1 resulted in epithelial morphogenesis failure, which was associated with a decrease in JNK activity. Collectively, our studies unprecedentedly elucidate JNK as the downstream mediator of the LKB1-dependent apoptosis, and provide a new paradigm for understanding the diverse LKB1 functions in vivo.

Nek6 overexpression antagonizes p53-induced senescence in human cancer cells.

Nek6 is an NIMA-related kinase that plays a critical role in mitotic cell cycle progression. Recent studies have shown that Nek6 is upregulated in various human cancers, but the function of Nek6 in tumorigenesis is largely unknown. Here, we examined the role of Nek6 in cellular senescence. Our data revealed that Nek6 expression is decreased both in both the replicative senescence of human normal fibroblasts and premature senescence induced by p53 expression in EJ human bladder cancer cells and H1299 human lung cancer cells. Interestingly, the enforced expression of Nek6 in EJ and H1299 cells completely suppresses p53-induced senescence, whereas the expression of kinase-dead Nek6 did not affect p53-induced senescence. Mechanistic studies revealed that cell cycle arrest in the G1 and G2/M phases, as well as the reduction of cyclin B and cdc2 protein level upon p53 expression were significantly reduced by Nek6 overexpression. In addition, p53-induced increases in intracellular levels of ROS were also inhibited in cells overexpressing Nek6. These results suggest that the downregulation of Nek6 expression is required for the onset of p53-induced cellular senescence and imply a possible role of Nek6 in tumorigenesis.

MKP-3 has essential roles as a negative regulator of the Ras/mitogen-activated protein kinase pathway during Drosophila development.

ABSTRACT Mitogen-activated protein kinase (MAPK) phosphatase 3 (MKP-3) is a well-known negative regulator in the Ras/extracellular signal-regulated kinase (ERK)-MAPK signaling pathway responsible for cell fate determination and proliferation during development. However, the physiological roles of MKP-3 and the mechanism by which MKP-3 regulates Ras/Drosophila ERK (DERK) signaling in vivo have not been determined. Here, we demonstrated that Drosophila MKP-3 (DMKP-3) is critically involved in cell differentiation, proliferation, and gene expression by suppressing the Ras/DERK pathway, specifically binding to DERK via the N-terminal ERK-binding domain of DMKP-3. Overexpression of DMKP-3 reduced the number of photoreceptor cells and inhibited wing vein differentiation. Conversely, DMKP-3 hypomorphic mutants exhibited extra photoreceptor cells and wing veins, and its null mutants showed striking phenotypes, such as embryonic lethality and severe defects in oogenesis. All of these phenotypes were highly similar to those of the gain-of-function mutants of DERK/rl. The functional interaction between DMKP-3 and the Ras/DERK pathway was further confirmed by genetic interactions between DMKP-3 loss-of-function mutants or overexpressing transgenic flies and various mutants of the Ras/DERK pathway. Collectively, these data provide the direct evidences that DMKP-3 is indispensable to the regulation of DERK signaling activity during Drosophila development.

Silent Information Regulator 2 (Sir2) and Forkhead box O (FOXO) Complement Mitochondrial Dysfunction and Dopaminergic Neuron Loss in Drosophila Pten-induced kinase 1 (PINK1) Null Mutant

Background: PINK1 loss of function induces mitochondrial dysfunction and dopaminergic neuron loss in Drosophila. Results: Sir2 shows a specific genetic interaction with PINK1 and rescues PINK1 null mutant phenotypes via FOXO. Conclusion: The strong genetic and functional interactions suggest that Sir2 and FOXO protect mitochondria and dopaminergic neuron downstream of PINK1. Significance: Understanding the molecular roles of PINK1 will be helpful for deciphering the molecular pathogenesis of Parkinson disease. PTEN-induced kinase 1 (PINK1), which is associated with early onset Parkinson disease, encodes a serine-threonine kinase that is critical for maintaining mitochondrial function. Moreover, another Parkinson disease-linked gene, parkin, functions downstream of PINK1 in protecting mitochondria and dopaminergic (DA) neuron. In our fly genetic screening, knockdown of Sir2 blocked PINK1 overexpression-induced phenotypes. Consistently, ectopic expression of Sir2 successfully rescued mitochondrial defects in PINK1 null mutants, but unexpectedly, failed in parkin mutants. In further genetic analyses, deletion of FOXO nullified the Sir2-induced mitochondrial restoration in PINK1 null mutants. Moreover, overexpression of FOXO or its downstream target gene such as SOD2 or Thor markedly ameliorated PINK1 loss-of-function defects, suggesting that FOXO mediates the mitochondrial protecting signal induced by Sir2. Consistent with its mitochondria-protecting role, Sir2 expression prevented the DA neuron loss of PINK1 null mutants in a FOXO-dependent manner. Loss of Sir2 or FOXO induced DA neuron degeneration, which is very similar to that of PINK1 null mutants. Furthermore, PINK1 deletion had no deleterious effect on the DA neuron loss in Sir2 or FOXO mutants, supporting the idea that Sir2, FOXO, and PINK1 protect DA neuron in a common pathway. Overall, these results strongly support the role of Sir2 and FOXO in preventing mitochondrial dysfunction and DA neuron loss, further suggesting that Sir2 and FOXO function downstream of PINK1 and independently of Parkin.