Cell cycle-dependent activation of Ras

AbstractBackground Ras proteins play an essential role in the transduction of signals from a wide range of cell-surface receptors to the nucleus. These signals may promote cellular proliferation or differentiation, depending on the cell background. It is well established that Ras plays an important role in the transduction of mitogenic signals from activated growth-factor receptors, leading to cell-cycle entry. However, important questions remain as to whether Ras controls signalling events during cell-cycle progression and, if so, at which point in the cell-cycle it is activated.Results To address these questions we have developed a novel, functional assay for the detection of cellular activated Ras. Using this assay, we found that Ras was activated in HeLa cells, following release from mitosis, and in NIH 3T3 fibroblasts, following serum-stimulated cell-cycle entry. In each case, peak Ras activation occurred in mid-G1 phase. Ras activation in HeLa cells at mid-G1 phase was dependent on RNA and protein synthesis and was not associated with tyrosine phosphorylation of Shc proteins and their binding to Grb2. Significantly, activation of Ras and the extracellular-signal regulated (ERK) subgroup of mitogen-activated protein kinases were not temporally correlated during G1-phase progression.Conclusions Activation of Ras during mid-G1 phase appears to differ in many respects from its rapid activation by growth factors, suggesting a novel mechanism of regulation that may be intrinsic to cell-cycle progression. Furthermore, the temporal dissociation between Ras and ERK activation suggests that Ras targets alternate effector pathways during G1-phase progression

An Active C-Terminally Truncated Form of Ca2+/Calmodulin-Dependent Protein Kinase Phosphatase-N (CaMKP-N/PPM1E)

Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) and its nuclear homolog CaMKP-N (PPM1E) are Ser/Thr protein phosphatases that belong to the PPM family. CaMKP-N is expressed in the brain and undergoes proteolytic processing to yield a C-terminally truncated form. The physiological significance of this processing, however, is not fully understood. Using a wheat-embryo cell-free protein expression system, we prepared human CaMKP-N (hCaMKP-N(WT)) and the truncated form, hCaMKP-N(1–559), to compare their enzymatic properties using a phosphopeptide substrate. The hCaMKP-N(1–559) exhibited a much higher value than the hCaMKP-N(WT) did, suggesting that the processing may be a regulatory mechanism to generate a more active species. The active form, hCaMKP-N(1–559), showed Mn2+ or Mg2+-dependent phosphatase activity with a strong preference for phospho-Thr residues and was severely inhibited by NaF, but not by okadaic acid, calyculin A, or 1-amino-8-naphthol-2,4-disulfonic acid, a specific inhibitor of CaMKP. It could bind to postsynaptic density and dephosphorylate the autophosphorylated Ca2+/calmodulin-dependent protein kinase II. Furthermore, it was inactivated by H2O2 treatment, and the inactivation was completely reversed by treatment with DTT, implying that this process is reversibly regulated by oxidation/reduction. The truncated CaMKP-N may play an important physiological role in neuronal cells.This work was supported, in part, by Grants-in-Aid for Scientific Research (21590334) from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant from the Japan Foundation for Applied Enzymology

Molecular mechanism of poly(ADP-ribosyl)ation by PARP1 and identification of lysine residues as ADP-ribose acceptor sites

Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR) using nicotinamide adenine dinucleotide (NAD) as a substrate. Despite intensive research on the cellular functions of PARP1, the molecular mechanism of PAR formation has not been comprehensively understood. In this study, we elucidate the molecular mechanisms of poly(ADP-ribosyl)ation and identify PAR acceptor sites. Generation of different chimera proteins revealed that the amino-terminal domains of PARP1, PARP2 and PARP3 cooperate tightly with their corresponding catalytic domains. The DNA-dependent interaction between the amino-terminal DNA-binding domain and the catalytic domain of PARP1 increased Vmax and decreased the Km for NAD. Furthermore, we show that glutamic acid residues in the auto-modification domain of PARP1 are not required for PAR formation. Instead, we identify individual lysine residues as acceptor sites for ADP-ribosylation. Together, our findings provide novel mechanistic insights into PAR synthesis with significant relevance for the different biological functions of PARP family members

Correction and lengthening for deformities of the forearm in multiple cartilaginous exostoses

金沢大学医学部附属病院整形外科Background. Multiple cartilaginous exostoses cause various deformities of the epiphysis. In exostoses of the ulna, the ulna is shortened and the radius acquires varus deformity, which may lead to dislocation of the radial head. In this study, we present the results of exostoses resection, with correction and lengthening with external fixators for functional and cosmetic improvement, and prevention of radial head dislocation. Methods. We retrospectively reviewed seven forearms of seven patients who had deformities of the forearm associated with multiple cartilaginous exostoses. One patient had dislocation of the radial head. Operative technique was excision of osteochondromas from the distal ulna, correction of the radius, and ulnar lengthening with external fixation up to 5 mm plus variance. We evaluated radiographs and the range of pronation and supination. Furthermore, we conducted a follow-up of ulnar length after the operation. Results. Dislocation of the radial head of one patient was naturally reduced without any operative intervention. At the most recent follow-up, six of the seven patients showed full improvement in pronation-supination. Ulnar shortening recurred with skeletal growth of four skeletally immature patients; however, it did not recur in one skeletally mature patient. Overlength of 5 mm was negated by the recurrence of ulnar shortening about 1.5 years after the operation. Conclusions. We treated seven forearms of seven patients by excision of osteochondromas, correction of radii, and gradual lengthening of ulnas with external fixators. The results of the procedure were satisfactory, especially for function of the elbow and wrist. However, we must consider the possible recurrence of ulnar shortening within about 1.5 years during skeletal growth periods in immature patients. © 2006 The Japanese Orthopaedic Association

A Kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury

© 2020, The Author(s). Renal tubular epithelial cells (RTECs) perform the essential function of maintaining the constancy of body fluid composition and volume. Toxic, inflammatory, or hypoxic-insults to RTECs can cause systemic fluid imbalance, electrolyte abnormalities and metabolic waste accumulation- manifesting as acute kidney injury (AKI), a common disorder associated with adverse long-term sequelae and high mortality. Here we report the results of a kinome-wide RNAi screen for cellular pathways involved in AKI-associated RTEC-dysfunction and cell death. Our screen and validation studies reveal an essential role of Cdkl5-kinase in RTEC cell death. In mouse models, genetic or pharmacological Cdkl5 inhibition mitigates nephrotoxic and ischemia-associated AKI. We propose that Cdkl5 is a stress-responsive kinase that promotes renal injury in part through phosphorylation-dependent suppression of pro-survival transcription regulator Sox9. These findings reveal a surprising non-neuronal function of Cdkl5, identify a pathogenic Cdkl5-Sox9 axis in epithelial cell-death, and support CDKL5 antagonism as a therapeutic approach for AKI

Protein phosphatases that regulate multifunctional Ca2+/calmodulin-dependent protein kinases: from biochemistry to pharmacology

Pergamon Press, Ishida, Atsuhiko ; Shigeri, Yasushi ; Taniguchi, Takanobu ; Kameshita, Isamu, Pharmacology and Therapeutics, 100(3), 2003, 291-305. authorMultifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs) play pivotal roles in Ca(2+) signaling pathways, such as the regulation of the neuronal functions of learning, memory, and neuronal cell death. The activities of the kinases are strictly regulated by protein phosphorylation/dephosphorylation. Although the activation mechanisms for multifunctional CaMKs through phosphorylation, which correspond to "switch on," have been extensively studied, the negative regulatory mechanisms through dephosphorylation, which correspond to "switch off," have not. In this review, we focused on the regulation of multifunctional CaMKs by the protein phosphatases responsible. We first summarized the current understanding of negative regulation of CaMKs by known protein phosphatases and their physiological significance. We then discussed newly developed methods for detection of protein phosphatases involved in the regulation of CaMKs. We also summarized the biochemical properties of a novel protein phosphatase, which we isolated with the new methods and designated as CaMK phosphatase (CaMKP), and its homologue. Pharmacological implications for neuronal functions including memory and neuronal cell death are discussed from the viewpoint that regulation of protein kinase activity can be elucidated by focusing on protein phosphatases involved in its "switch off" mechanism