phosphothreonine and Neoplasms

The prolyl isomerase Pin1 is a conserved enzyme that is intimately involved in diverse biological processes and pathological conditions such as cancer and Alzheimer's disease. By catalysing cis-trans interconversion of certain motifs containing phosphorylated serine or threonine residues followed by a proline residue (pSer/Thr-Pro), Pin1 can have profound effects on phosphorylation signalling. The structural and functional differences that result from cis-trans isomerization of specific pSer/Thr-Pro motifs probably underlie most, if not all, Pin1-dependent actions. Phosphorylation-dependent prolyl isomerization by Pin1 remains a unique mode for the modulation of signal transduction. Here, we provide an overview of the plethora of regulatory events that involve this unique enzyme, with a particular focus on oncogenic signalling and neurodegeneration.

Topics: Alzheimer Disease; Cell Proliferation; Humans; Isomerism; Neoplasms; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Conformation; Signal Transduction

Protein phosphorylation on certain serine or threonine residues preceding proline (Ser/Thr-Pro) is a pivitol signaling mechanism in diverse cellular processes and its deregulation can lead to human disease. However, little is known about how these phosphorylation events actually control cell signaling. Pin1 is a highly conserved enzyme that isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Recent results indicate that such conformational changes following phosphorylation are a novel signaling mechanism pivotal in regulating many cellular functions. This mechanism also offers new insights into the pathogenesis and treatment of human disease, most notably cancer and Alzheimer's disease. Thus, Pin1 plays a key role in linking signal transduction to the pathogenesis of cancer and Alzheimer's disease - two major age-related diseases.

Topics: Alzheimer Disease; CDC2-CDC28 Kinases; Cell Cycle; Cyclin-Dependent Kinase 2; Humans; Models, Biological; Models, Molecular; Neoplasms; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Phosphoserine; Phosphothreonine; Proline; Proline-Directed Protein Kinases; Protein Structure, Tertiary; Signal Transduction

Protein p130Cas constitutes an adaptor protein mainly involved in integrin signaling downstream of Src kinase. Owing to its modular structure, p130Cas acts as a general regulator of cancer cell growth and invasiveness induced by different oncogenes. However, other mechanisms of p130Cas signaling leading to malignant progression are poorly understood. Here, we show a novel interaction of p130Cas with Ser/Thr kinase PKN3, which is implicated in prostate and breast cancer growth downstream of phosphoinositide 3-kinase. This direct interaction is mediated by the p130Cas SH3 domain and the centrally located PKN3 polyproline sequence. PKN3 is the first identified Ser/Thr kinase to bind and phosphorylate p130Cas and to colocalize with p130Cas in cell structures that have a pro-invasive function. Moreover, the PKN3-p130Cas interaction is important for mouse embryonic fibroblast growth and invasiveness independent of Src transformation, indicating a mechanism distinct from that previously characterized for p130Cas. Together, our results suggest that the PKN3-p130Cas complex represents an attractive therapeutic target in late-stage malignancies.

Topics: Animals; Cell Movement; Cell Proliferation; Crk-Associated Substrate Protein; Fibroblasts; Humans; Mice, Nude; Neoplasm Invasiveness; Neoplasms; Phosphorylation; Phosphothreonine; Podosomes; Protein Binding; Protein Kinase C; Pseudopodia; src-Family Kinases; Stress Fibers

Akt, also known as protein kinase B, plays key roles in cell proliferation, survival and metabolism. Akt hyperactivation contributes to many pathophysiological conditions, including human cancers, and is closely associated with poor prognosis and chemo- or radiotherapeutic resistance. Phosphorylation of Akt at S473 (ref. 5) and T308 (ref. 6) activates Akt. However, it remains unclear whether further mechanisms account for full Akt activation, and whether Akt hyperactivation is linked to misregulated cell cycle progression, another cancer hallmark. Here we report that Akt activity fluctuates across the cell cycle, mirroring cyclin A expression. Mechanistically, phosphorylation of S477 and T479 at the Akt extreme carboxy terminus by cyclin-dependent kinase 2 (Cdk2)/cyclin A or mTORC2, under distinct physiological conditions, promotes Akt activation through facilitating, or functionally compensating for, S473 phosphorylation. Furthermore, deletion of the cyclin A2 allele in the mouse olfactory bulb leads to reduced S477/T479 phosphorylation and elevated cellular apoptosis. Notably, cyclin A2-deletion-induced cellular apoptosis in mouse embryonic stem cells is partly rescued by S477D/T479E-Akt1, supporting a physiological role for cyclin A2 in governing Akt activation. Together, the results of our study show Akt S477/T479 phosphorylation to be an essential layer of the Akt activation mechanism to regulate its physiological functions, thereby providing a new mechanistic link between aberrant cell cycle progression and Akt hyperactivation in cancer.

Topics: Animals; Apoptosis; Cell Cycle; Cell Proliferation; Cyclin A2; Cyclin-Dependent Kinase 2; Embryonic Stem Cells; Enzyme Activation; Male; Mechanistic Target of Rapamycin Complex 2; Mice; Multiprotein Complexes; Neoplasms; Olfactory Bulb; Oncogene Protein v-akt; Phosphorylation; Phosphoserine; Phosphothreonine; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases

Eukaryotic elongation factor 2 kinase (eEF2K) is the best-characterized member of the α-kinase family. Within this group, only eEF2K and myosin heavy chain kinases (MHCKs) have known substrates. Here we have studied the roles of specific residues, selected on the basis of structural data for MHCK A and TRPM7, in the function of eEF2K. Our data provide the first information regarding the basis of the substrate specificity of α-kinases, in particular the roles of residues in the so-called N/D loop, which appears to occupy a position in the structure of α-kinases similar to that of the activation loop in other kinases. Several mutations in the EEF2K gene occur in tumors, one of which (Arg303Cys) is at a highly conserved residue in the N/D loop. This mutation greatly enhances eEF2K activity and may be cytoprotective. Our data support the concept that the major autophosphorylation site (Thr348 in eEF2K) docks into a binding pocket to help create the kinase-competent conformation. This is similar to the situation for MHCK A and is consistent with this being a common feature of α-kinases.

Topics: Amino Acid Sequence; Amino Acids; Binding Sites; Calcium-Calmodulin-Dependent Protein Kinases; Catalytic Domain; Conserved Sequence; Elongation Factor 2 Kinase; HEK293 Cells; Humans; Models, Molecular; Molecular Sequence Data; Mutation; Neoplasms; Phosphorylation; Phosphothreonine; Protein Binding; Protein Structure, Secondary; Protozoan Proteins; Structural Homology, Protein; Structure-Activity Relationship; Substrate Specificity

Tumor-derived microvesicles are rich in metastasis-related proteases and play a role in the interactions between tumor cells and tumor microenvironment in tumor metastasis. Because shed microvesicles may remain in the extracellular environment around tumor cells, the microvesicle membrane protein may be the potential target for cancer therapy. Here we report that chromosome segregation 1-like (CSE1L) protein is a microvesicle membrane protein and is a potential target for cancer therapy. v-H-Ras expression induced extracellular signal-regulated kinase (ERK)-dependent CSE1L phosphorylation and microvesicle biogenesis in various cancer cells. CSE1L overexpression also triggered microvesicle generation, and CSE1L knockdown diminished v-H-Ras-induced microvesicle generation, matrix metalloproteinase (MMP)-2 and MMP-9 secretion and metastasis of B16F10 melanoma cells. CSE1L was preferentially accumulated in microvesicles and was located in the microvesicle membrane. Furthermore, anti-CSE1L antibody-conjugated quantum dots could target tumors in animal models. Our findings highlight a novel role of Ras-ERK signaling in tumor progression and suggest that CSE1L may be involved in the "early" and "late" metastasis of tumor cells in tumorigenesis. Furthermore, the novel microvesicle membrane protein, CSE1L, may have clinical utility in cancer diagnosis and targeted cancer therapy.

Topics: Animals; Antibodies; Cell Line, Tumor; Cell-Derived Microparticles; Cellular Apoptosis Susceptibility Protein; Extracellular Signal-Regulated MAP Kinases; Humans; Male; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasms; Phosphorylation; Phosphothreonine; ras Proteins

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an important member of the tumor necrosis factor subfamily with great potential in cancer therapy. Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess potent anti-inflammatory and anticancer activities. Here, we showed that pretreatment with Andro significantly enhances TRAIL-induced apoptosis in various human cancer cell lines, including those TRAIL-resistant cells. Such sensitization is achieved through transcriptional up-regulation of death receptor 4 (DR4), a death receptor of TRAIL. In search of the molecular mechanisms responsible for DR4 up-regulation, we found that the tumor suppressor p53 plays an essential role in DR4 transcriptional activation. Andro is capable of activating p53 via increased p53 phosphorylation and protein stabilization, a process mediated by enhanced reactive oxygen species production and subsequent c-Jun NH(2)-terminal kinase activation. Pretreatment with an antioxidant (N-acetylcysteine) or a c-Jun NH(2)-terminal kinase inhibitor (SP600125) effectively prevented Andro-induced p53 activation and DR4 up-regulation and eventually blocked the Andro-induced sensitization on TRAIL-induced apoptosis. Taken together, these results present a novel anticancer effect of Andro and support its potential application in cancer therapy to overcome TRAIL resistance.

Topics: Apoptosis; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspases; Cell Line, Tumor; Diterpenes; Down-Regulation; Drug Screening Assays, Antitumor; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Neoplasms; Phosphorylation; Phosphothreonine; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; TNF-Related Apoptosis-Inducing Ligand; Transcription, Genetic; Tumor Suppressor Protein p53; Up-Regulation; X-Linked Inhibitor of Apoptosis Protein

Earlier studies have demonstrated a functional link between B56gamma-specific protein phosphatase 2A (B56gamma-PP2A) and p53 tumor suppressor activity. Upon DNA damage, a complex including B56gamma-PP2A and p53 is formed which leads to Thr55 dephosphorylation of p53, induction of the p53 transcriptional target p21, and the inhibition of cell proliferation. Although an enhanced interaction between p53 and B56gamma is observed after DNA damage, the underlying mechanism and its significance in PP2A tumor-suppressive function remain unclear. In this study, we show that the increased interaction between B56gamma and p53 after DNA damage requires ATM-dependent phosphorylation of p53 at Ser15. In addition, we demonstrate that the B56gamma3-induced inhibition of cell proliferation, induction of cell cycle arrest in G(1), and blockage of anchorage-independent growth are also dependent on Ser15 phosphorylation of p53 and p53-B56gamma interaction. Taken together, our results provide a mechanistic link between Ser15 phosphorylation-mediated p53-B56gamma interaction and the modulation of p53 tumor suppressor activity by PP2A. We also show an important link between ATM activity and the tumor-suppressive function of B56gamma-PP2A.

Topics: Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; DNA Damage; DNA-Binding Proteins; Humans; Mutation; Neoplasms; Phosphoserine; Phosphothreonine; Protein Binding; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The Cdc25A phosphatase positively regulates cell-cycle transitions, is degraded by the proteosome throughout interphase and in response to stress, and is overproduced in human cancers. The kinases targeting Cdc25A for proteolysis during early cell-cycle phases have not been identified, and mechanistic insight into the cause of Cdc25A overproduction in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3beta (GSK-3beta) phosphorylates Cdc25A to promote its proteolysis in early cell-cycle phases. Phosphorylation by GSK-3beta requires priming of Cdc25A, and this can be catalyzed by polo-like kinase 3 (Plk-3). Importantly, a strong correlation between Cdc25A overproduction and GSK-3beta inactivation was observed in human tumor tissues, indicating that GSK-3beta inactivation may account for Cdc25A overproduction in a subset of human tumors.

Topics: Animals; beta-Transducin Repeat-Containing Proteins; cdc25 Phosphatases; Cell Cycle; Cell Line, Tumor; Checkpoint Kinase 1; Enzyme Activation; Enzyme Stability; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Mice; Models, Biological; Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Binding; Protein Kinases; Protein Processing, Post-Translational; Radiation, Ionizing; Ubiquitin

The transcription factor Sp1 is ubiquitously expressed in different cells and thereby regulates the expression of genes involved in many cellular processes. This study reveals that Sp1 was phosphorylated during the mitotic stage in three epithelial tumor cell lines and one glioma cell line. By using different kinase inhibitors, we found that during mitosis in HeLa cells, the c-Jun NH(2)-terminal kinase (JNK) 1 was activated that was then required for the phosphorylation of Sp1. In addition, blockade of the Sp1 phosphorylation via inhibition JNK1 activity in mitosis resulted in the ubiquitination and degradation of Sp1. JNK1 phosphorylated Sp1 at Thr278/739. The Sp1 mutated at Thr278/739 was unstable during mitosis, possessing less transcriptional activity for the 12(S)-lipoxygenase expression and exhibiting a decreased cell growth rate compared with wild-type Sp1 in HeLa cells. In N-methyl-N-nitrosourea-induced mammary tumors, JNK1 activation provided a potential relevance with the accumulation of Sp1. Together, our results indicate that JNK1 activation is necessary to phosphorylate Sp1 and to shield Sp1 from the ubiquitin-dependent degradation pathway during mitosis in tumor cell lines.

Topics: Animals; Arachidonate 12-Lipoxygenase; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Female; Humans; Methylnitrosourea; Mitogen-Activated Protein Kinase 8; Mitosis; Mutant Proteins; Neoplasms; Phosphorylation; Phosphothreonine; Protein Transport; Rats; Rats, Sprague-Dawley; Sp1 Transcription Factor; Thermodynamics; Transcription, Genetic

Polymerized type I collagen suppresses fibroblast proliferation. Previous studies have implicated inhibition of fibroblast proliferation with polymerized collagen-mediated suppression of S6K1, but the molecular mechanism of the critical negative feedback loop has not yet been fully elucidated. Here, we demonstrate that polymerized collagen suppresses G(1)/S phase transition and fibroblast proliferation by a novel mechanism involving the formation of a beta1 integrin-protein phosphatase 2A (PP2A)-tuberous sclerosis complex 2 (TSC2) complex that represses S6K1 activity. In response to fibroblast interaction with polymerized collagen, beta1 integrin forms a complex with PP2A that targets TSC2 as a substrate. PP2A represses the level of TSC2 phosphorylation and maintains TSC2 in an activated state. Activated TSC2 negatively regulates the downstream kinase S6K1 and inhibits G(1)/S transit. Knockdown of TSC2 enables fibroblasts to overcome the anti-proliferative properties of polymerized collagen. Furthermore, we show that this reduction in TSC2 and S6K1 phosphorylation occurs largely independent of Akt. Although S6K1 activity was markedly suppressed by polymerized collagen, we found that minimal changes in Akt activity occurred. We demonstrate that up-regulation of Akt by overexpression of constitutively active phosphatidylinositol 3-kinase p110 subunit had minor effects on TSC2 and S6K1 phosphorylation. These findings demonstrate that polymerized collagen represses fibroblast proliferation by a mechanism involving the formation of a beta1 integrin-PP2A-TSC2 complex that negatively regulates S6K1 and inhibits G(1)/S phase transition.

Topics: Cell Adhesion; Cell Line; Cell Proliferation; Collagen; Enzyme Activation; Fibroblasts; Humans; Integrin beta1; Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoserine; Phosphothreonine; Protein Binding; Protein Phosphatase 2; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases; RNA, Small Interfering; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Growth factor-dependent accumulation of the cyclin D1 proto-oncogene is balanced by its rapid phosphorylation-dependent proteolysis. Degradation is triggered by threonine 286 phosphorylation, which promotes its ubiquitination by an unknown E3 ligase. We demonstrate that Thr286-phosphorylated cyclin D1 is recognized by a Skp1-Cul1-F box (SCF) ubiquitin ligase where FBX4 and alphaB crystallin govern substrate specificity. Overexpression of FBX4 and alphaB crystallin triggered cyclin D1 ubiquitination and increased cyclin D1 turnover. Impairment of SCF(FBX4-alphaB crystallin) function attenuated cyclin D1 ubiquitination, promoting cyclin D1 overexpression and accelerated cell-cycle progression. Purified SCF(FBX4-alphaB crystallin) catalyzed polyubiquitination of cyclin D1 in vitro. Consistent with a putative role for a cyclin D1 E3 ligase in tumorigenesis, FBX4 and alphaB crystallin expression was reduced in tumor-derived cell lines and a subset of primary human cancers that overexpress cyclin D1. We conclude that SCF(FBX4-alphaB crystallin) is an E3 ubiquitin ligase that promotes ubiquitin-dependent degradation of Thr286-phosphorylated cyclin D1.

Topics: alpha-Crystallin B Chain; Animals; Catalysis; Cyclin D1; Cytoplasm; F-Box Proteins; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Mice; Neoplasms; NIH 3T3 Cells; Phosphorylation; Phosphothreonine; Protein Binding; Protein Transport; Proto-Oncogene Mas; RNA, Messenger; SKP Cullin F-Box Protein Ligases; Thermodynamics; Ubiquitin