scholarly journals RASA3 Regulates Stage-Specific AKT Signaling and Cell Cycle Progression in Mammalian Erythropoiesis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-3
Author(s):  
Elena C. Brindley ◽  
Emily Hartman ◽  
Julien Papoin ◽  
Jeffrey Michael Lipton ◽  
Luanne L. Peters ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Cycle Progression ◽  
Bone Marrow Failure ◽  
Erythroid Differentiation ◽  
Akt Signaling ◽  
Ras Signaling ◽  
Loss Of Function ◽  
Cycle Progression

Inherited bone marrow failure syndromes (IBMFS) are a heterogenous group of disorders characterized by dysregulated hematopoiesis across various lineages, predisposition to malignancy, and diverse syndromic features. The scat (severe combined anemia and thrombocytopenia) mouse model has been characterized as a unique model of IBMFS. Scat carries an autosomal recessive missense mutation in the Rasa3 gene that results in RASA3 mislocalization and loss of function. RASA3 functions as a Ras-GTPase activating protein, and its loss of function in scat results in increased erythroid Ras activity, increased reactive oxygen species, and altered cell cycle progression, all culminating in delayed terminal erythroid differentiation. However, the precise mechanism of RASA3 regulation of erythroid differentiation remains undefined, and elucidation of this mechanism is crucial to identifying new therapeutic targets in inherited anemia. Considering the role of RASA3 as regulator of Ras signaling and cell cycle progression, and the importance of these processes to erythroid differentiation, we sought to first characterize the coordination of Ras signaling pathways and cell cycle progression in normal murine erythropoiesis, then observe how loss of RASA3 function alters this regulatory axis. We observed that wild type (WT) erythroblasts demonstrate population-specific influence of ERK and PI3K/AKT signaling in regulating cell cycle progression. Inhibition of both pathways with increasing doses of U0126 and LY294002, respectively, induced accumulation in G0/G1 from the proerythroblast stage until the late basophilic/polychromatic stage (U0126 vehicle vs. 1uM p= 0.0023; LY294002 vehicle vs 1uM p=0.0389). At these later stages, ERK and PI3K/AKT inhibition led to a decrease in G0/G1 percentages, suggesting a stage-specific switch in signaling mediated cell cycle regulation, with PI3K inhibition demonstrating more potent and consistent effects (U0126 vehicle vs 1uM p=0.0406, 0.0481; LY294002 vehicle vs 1uM p=0.0003, 0.0197, 0.0086, n=3). These patterns suggest that ERK and AKT may facilitate cell cycle progression past the G0/G1 checkpoint in early erythropoiesis while inducing cell cycle exit or accumulation in G0/G1 in late erythropoiesis. In scat, we previously characterized increased active Ras in erythroid cells and a delay in terminal erythroid differentiation with accumulation at the polychromatic stage. We therefore next sought to examine the potential mechanistic contribution of altered PI3K/AKT signaling and cell cycle progression to the differentiation delay seen in scat. Phospho-flow analyses demonstrate that scat bone marrow-derived basophilic and polychromatic erythroblasts have increased AKT activation compared to WT (p=0.0402, p=0.0559, respectively; n=4), with similar trends evident in scat spleen basophilic erythroblasts (p=0.064; n=4). These results are consistent with increased Ras activation in scat. Ex vivo EdU/PI analyses revealed that scat bone marrow-derived polychromatic erythroblasts demonstrate G0/G1 accumulation (p=0.0466) and decreased progression to S-phase (0.0414; n=6), also with similar trends in scat spleen basophilic erythroblasts (p=0.004; n=6). These results correlate with the observed differentiation delay in scat and indicate that RASA3 regulates stage-specific signaling and cell cycle progression during erythropoiesis. To study if cell cycle dysregulation in scat begins at an earlier stage of erythroid differentiation, we analyzed murine hematopoietic and erythroid progenitors as Ter119-, cKit+ cells expressing increasing levels of CD71 and found that both CD71lo and CD71med bone marrow-derived scat progenitors present with G0/G1 accumulation (p=0.0015, p=0.0073, respectively) and decreased progression to S-phase (p=0.0014, p=0.0241; n=7). This suggests a dynamic relationship between RASA3, Ras signaling, and cell cycle progression throughout early and late erythroid differentiation. Together, these findings support the role of RASA3 as a regulator of the signaling networks governing erythropoiesis and reveal a new targetable axis in a model of inherited bone marrow failure. Disclosures No relevant conflicts of interest to declare.

scholarly journals RASA3 Deficiency Contributes to Anemia By Multiple Mechanisms

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 920-920
Author(s):  
Elena C. Brindley ◽  
Emily Hartman ◽  
Julien Papoin ◽  
Brian Dulmovits ◽  
Steven L. Ciciotte ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Cycle Progression ◽  
Bone Marrow Failure ◽  
Erythroid Differentiation ◽  
Ras Signaling ◽  
Cycle Progression ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Terminal Erythroid Differentiation

Abstract RASA3, a Ras GTPase activating protein, is critical to vertebrate erythropoiesis and megakaryopoiesis. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a G125V mutation in Rasa3 that leads to profound bone marrow failure with characteristics of aplastic anemia. The phenotype is cyclic, and mice alternate between periods of crisis and remission. Our previous studies demonstrated that this mutation in Rasa3 causes defects in several aspects of erythropoiesis, including a significant delay of erythroid differentiation at the polychromatophilic stage, decreased hemoglobinization, defects in cell cycle progression past the G1 checkpoint, and increased reactive oxygen species (ROS) during terminal erythroid differentiation as well as in scat peripheral blood reticulocytes and red blood cells. We previously reported that the mislocalization of mutated RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the erythropoietic defect in scat, and the observed cell cycle arrest and increased ROS likely also contribute to this unique disease phenotype. Our current efforts are focused on further elucidation of the mechanism and specific disruptions in Ras signaling that lead to anemia, membrane fragmentation, and the cyclic phenotype in scat. Interestingly, we report here that apoptosis is not increased during scat crisis, and that mitochondria, a potential source of ROS, are normally eliminated at the reticulocyte stage. The dramatic nature of remission, with complete normalization of all hematologic parameters, led us to hypothesize that a secreted factor may be mediating the cyclic phenotype of scat. Differences in the cytokine profile of the serum of scat mice compared to wild type suggest that, indeed, one or several secreted factor(s) may be influencing the occurrence of bone marrow failure. Levels of galectin-1, a known mediator of cell-cell interactions and intracellular signaling in the hematopoietic niche, are consistently decreased in scat serum according to a multispot anti-cytokine antibody array (23,326.5 ± 21,439.7 integrated density in scat vs. 31,019.6± 20,110.7 in controls, p<0.05).Studies exploring the influence of the galectin family on erythropoiesis and Ras signaling in the context of scat are underway. Strengthening the notion that RASA3 has a critical conserved role in vertebrate terminal erythropoiesis, the characteristics of bone marrow failure seen in scat have been reproduced in human CD34+ cells using siRNA and shRNA knockdowns of Rasa3 . Similar to the changes seen in scat, cells with decreased RASA3 demonstrated delayed terminal erythroid differentiation and defective hemoglobinization. Finally, analysis of Ras expression and functional pull-down studies in human CD34+ cells revealed that, while K-Ras is the major active isoform expressed during terminal erythroid differentiation, H-Ras is also active during human erythropoiesis. Future studies with CD34+ Rasa3 knockdown cells will explore the influence of RASA3 on human K- and H-Ras signaling in erythropoiesis. Taken together, our studies further characterize the vital role of RASA3 in hemoglobinization, cell cycle progression, and cell survival during terminal erythroid differentiation, as well as identify novel targets for investigation of unknown mechanisms (e.g., dysregulated cytokine secretion) of bone marrow failure syndromes. Disclosures No relevant conflicts of interest to declare.

scholarly journals RASA3 Is Involved in Cell Cycle Progression, Hemoglobinization and Generation of Reactive Oxygen Species during Mammalian Erythropoiesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3328-3328
Author(s):  
Brian M. Dulmovits ◽  
Yue Zhao ◽  
Luanne L. Peters ◽  
Lionel Blanc
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Cycle Progression ◽  
Bone Marrow Failure ◽  
Erythroid Differentiation ◽  
Oxygen Species ◽  
Cycle Progression ◽  
Cd34 Cells

Abstract RASA3, a Ras GTPase activating protein, is critical to vertebrate erythropoiesis and megakaryopoiesis. Defective RASA3 in zebrafish and mice results in severe anemia and thrombocytopenia. Indeed, in the mouse model scat (severe combined anemia and thrombocytopenia), a G125V mutation in Rasa3 leads to profound bone marrow failure with characteristics of aplastic anemia. The phenotype is cyclic, and mice alternate between periods of crisis and remission. Previous studies showed that this mutation in Rasa3 causes severely delayed erythroid differentiation at the polychromatophilic stage and decreased hemoglobinization due, at least in part, to mislocalization of RASA3 to the cytosol and resultant increased Ras activity. Here, we provide evidence that RASA3 plays a pivotal role in cell cycle progression and maintenance of reactive oxygen species (ROS) levels in erythropoiesis during crisis episodes. First, we analyzed the cell cycle progression in scat erythroblasts during crisis episodes. Using propidium iodide and flow cytometry, we found a significant increase in the G0/G1 phase (46.8% ± 3.1% in crisis vs 34.8% ± 2.5 in controls, p<0.001) while S phase was decreased (40.2% ± 2.9% vs. 49.6% ± 3.3% p<0.01) and G2 was not affected in scat proerythroblasts. These data suggest that Rasa3 is involved in the G1 checkpoint. In addition, we observed an increase in the ROS levels in scat throughout differentiation including proerythroblasts (1.03% ± 0.3% vs. 0.25% ± 0.1% in controls, p < 0.01), EryA (1.5% ± 0.21% vs. 0.25% ± 0.05%, p < 0.01), EryB (2.6% ± 1.41% vs. 0.25% ± 0.01%, p < 0.01), EryC (2.0% ± 0.45% vs. 0.25% ± 0.02%, p < 0.01) and mature red cells (1.15% ± 0.1% vs. 0.9% ± 0.03%, p < 0.05); the highest levels of ROS were observed in the basophilic-polychromatic erythroblast (i.e. EryB) containing populations. In addition, we consistently found increased levels of ROS in both reticulocytes and red blood cells from scat peripheral blood. Therefore, the cell cycle arrest and increased ROS likely contribute to the erythropoietic defect associated with scat. No mutations involving RASA3 have been involved in human bone marrow failure syndromes yet. However, in more than 30% of the cases, the etiology remains unexplained. To understand the putative role of RASA3 in the pathophysiology of hematopoiesis, and based on our data obtained in the mouse and the zebrafish, we investigated the role of RASA3 in human erythropoiesis using si and shRNA knockdowns in cord blood-derived CD34+ cells. Knockdown efficiencies were evaluated by western blot, and revealed that RASA3 protein levels were reduced by greater than 50% using two different shRNA constructs and siRNA. Cells were differentiated using an adapted 3-phase liquid culture system that fully recapitulates erythropoiesis. Similar to the scat mouse, we observed delayed differentiation by flow cytometry using glycophorin A, band3 and α4-integrin as markers of terminal differentiation with 62.0% of RASA3 knockdown erythroblasts α4-integrinhi (compared to 27.5% in controls) at day 16. In addition, the nucleus was substantially less pyknotic and the nucleocytoplasmic ratio remained elevated during differentiation, as compared to control cells. Indeed, 64 ± 7% of the cells had an uncondensed nucleus in the RASA3 knockdown samples, compared to 30 ± 3% in the scramble controls. Moreover as in scat, hemoglobinization was defective; qRT-PCR analyses revealed a 25% reduction in γ-globin and a 20% reduction in β-globin expression. These findings strengthen and emphasize the notion that RASA3 has a conserved function during vertebrate terminal erythropoiesis. Experiments investigating cell cycle progression, apoptosis and ROS production in human CD34+ cells are underway. Together, our studies demonstrate that RASA3 plays and important role in hemoglobinization, cell cycle progression and cell survival during terminal erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Essential Role of p400/mDomino Chromatin-remodeling ATPase in Bone Marrow Hematopoiesis and Cell-cycle Progression

2010 ◽  
Vol 285 (39) ◽  
pp. 30214-30223 ◽  
Author(s):  
Toshihiro Fujii ◽  
Takeshi Ueda ◽  
Shigekazu Nagata ◽  
Rikiro Fukunaga
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Chromatin Remodeling ◽  
Cell Cycle Progression ◽  
Essential Role ◽  
Cycle Progression

CD81 Is Essential for HSC Self-Renewal through Suppressing Proliferation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 76-76 ◽  
Author(s):  
Kuanyin Karen Lin ◽  
Lara Rossi ◽  
Margaret A. Goodell
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Cycle Progression ◽  
Background Level ◽  
Quiescent State ◽  
Self Renewal ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Transplantation Assay

Abstract Hematopoietic stem cells (HSCs) comprise only ~0.02% of the whole bone marrow cells but possess the capacity to extensively proliferate in order to restore hematopoietic homeostasis. Under homeostasis, HSCs are relatively quiescent with a slow cell cycle progression rate. However, upon stimulation, HSCs are able to promptly proliferate and undergo self-renewal to regenerate HSCs as daughter cells. While regulatory mechanisms involved in cell cycle progression are well characterized to be essential for HSC self-renewal, the mechanisms that facilitate the return of proliferating HSC to their quiescent state have been largely overlooked. The expression of CD81 (also called TAPA-1), a transmembrane protein that belongs to the Tetraspanin family, has been found associated with HSC proliferation. While CD81 is normally absent on HSC, it becomes markedly upregulated during HSC proliferation (Figure 1). To understand the function of CD81 in regenerating HSCs, we utilized a murine stem cell retroviral vector to deliver genes into 5-FU treated bone marrow progenitors to test the effect of enforced CD81 overexpression on HSC. The CD81-transduced proliferating progenitors were found to give rise to an increased number of phenotypically-defined HSC (SP-KLS) without significantly affecting the homeostasis in peripheral organs. In addition, we also characterized the HSCs from CD81 knock-out mice. We discovered that CD81-null HSC failed to engraft in peripheral blood of secondary recipients in serial transplantation assays (Figure 2), suggesting a role of CD81 in preserving a functional HSC compartment during proliferation-induced stress. When investigating further, we discovered that CD81 is a cell cycle suppressor for HSC, as the CD81KO HSCs are delayed in returning quiescence. In addition, clustering of CD81 on the HSC cell membrane using a monoclonal antibody rapidly induced a quiescent phenotype. This was found to be associated with an altered phosphorylation level of AKT, an inhibitor of the transcription factor FOXO1a and FOXO3a, which have been reported to be essential for HSC self-renewal through suppressing HSC proliferation. Taken together, these results demonstrate an essential role of CD81 in HSC self-renewal, and a novel mechanism that advances quiescence from a proliferating state. Figure 1. CD81 expression is upregulated at the time when HSCs (SPKLS) are proliferating in response to 5FU stimulation, a chemotheraputic agent that induces HSC to proliferate. The expression of CD81 is found at a background level in quiescent stages (5FU-Day0 and 5FU-Day11), and is upregulated during proliferation stages (starting 5FU-Day2) Figure 1. CD81 expression is upregulated at the time when HSCs (SPKLS) are proliferating in response to 5FU stimulation, a chemotheraputic agent that induces HSC to proliferate. The expression of CD81 is found at a background level in quiescent stages (5FU-Day0 and 5FU-Day11), and is upregulated during proliferation stages (starting 5FU-Day2) Figure 2. CD8KO HSCs fail to engraft in the secondary competitive transplantation assay, indicating a self-renewal defect. In this assay, 300 donor-derived HSCs (CD45.2 SPKLS) were purified from the primary recipients and transplanted along with 2×105 competitors into lethally irradiated mice (**p<0.01). Figure 2. CD8KO HSCs fail to engraft in the secondary competitive transplantation assay, indicating a self-renewal defect. In this assay, 300 donor-derived HSCs (CD45.2 SPKLS) were purified from the primary recipients and transplanted along with 2×105 competitors into lethally irradiated mice (**p<0.01).

scholarly journals ROR2 has a protective role in melanoma by inhibiting Akt activity, cell-cycle progression, and proliferation

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
María Victoria Castro ◽  
Gastón Alexis Barbero ◽  
María Belén Villanueva ◽  
Luca Grumolato ◽  
Jérémie Nsengimana ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Growth ◽  
Cell Cycle Progression ◽  
Orphan Receptor ◽  
Cell Cycle Regulators ◽  
Loss Of Function ◽  
Proliferation Markers ◽  
Cycle Progression ◽  
Primary Tumors

Abstract Background Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a Wnt5a receptor aberrantly expressed in cancer that was shown to either suppress or promote carcinogenesis in different tumor types. Our goal was to study the role of ROR2 in melanoma. Methods Gain and loss-of-function strategies were applied to study the biological function of ROR2 in melanoma. Proliferation assays, flow cytometry, and western blotting were used to evaluate cell proliferation and changes in expression levels of cell-cycle and proliferation markers. The role of ROR2 in tumor growth was assessed in xenotransplantation experiments followed by immunohistochemistry analysis of the tumors. The role of ROR2 in melanoma patients was assessed by analysis of clinical data from the Leeds Melanoma Cohort. Results Unlike previous findings describing ROR2 as an oncogene in melanoma, we describe that ROR2 prevents tumor growth by inhibiting cell-cycle progression and the proliferation of melanoma cells. The effect of ROR2 is mediated by inhibition of Akt phosphorylation and activity which, in turn, regulates the expression, phosphorylation, and localization of major cell-cycle regulators including cyclins (A, B, D, and E), CDK1, CDK4, RB, p21, and p27. Xenotransplantation experiments demonstrated that ROR2 also reduces proliferation in vivo, resulting in inhibition of tumor growth. In agreement with these findings, a higher ROR2 level favors thin and non-ulcerated primary melanomas with reduced mitotic rate and better prognosis. Conclusion We conclude that the expression of ROR2 slows down the growth of primary tumors and contributes to prolonging melanoma survival. Our results demonstrate that ROR2 has a far more complex role than originally described.

PTTG has a Dual Role of Promotion-Inhibition in the Development of Pituitary Adenomas

2019 ◽  
Vol 26 (11) ◽  
pp. 800-818
Author(s):  
Zujian Xiong ◽  
Xuejun Li ◽  
Qi Yang
Keyword(s):  
Cell Cycle ◽  
Pituitary Adenoma ◽  
Pituitary Adenomas ◽  
Cell Cycle Progression ◽  
Key Factors ◽  
Cycle Progression ◽  
Sister Chromatids ◽  
Regulation Of Cell Cycle ◽  
Late Stages

Pituitary Tumor Transforming Gene (PTTG) of human is known as a checkpoint gene in the middle and late stages of mitosis, and is also a proto-oncogene that promotes cell cycle progression. In the nucleus, PTTG works as securin in controlling the mid-term segregation of sister chromatids. Overexpression of PTTG, entering the nucleus with the help of PBF in pituitary adenomas, participates in the regulation of cell cycle, interferes with DNA repair, induces genetic instability, transactivates FGF-2 and VEGF and promotes angiogenesis and tumor invasion. Simultaneously, overexpression of PTTG induces tumor cell senescence through the DNA damage pathway, making pituitary adenoma possessing the potential self-limiting ability. To elucidate the mechanism of PTTG in the regulation of pituitary adenomas, we focus on both the positive and negative function of PTTG and find out key factors interacted with PTTG in pituitary adenomas. Furthermore, we discuss other possible mechanisms correlate with PTTG in pituitary adenoma initiation and development and the potential value of PTTG in clinical treatment.

scholarly journals Phosphorylation of RCC1 on Serine 11 Facilitates G1/S Transition in HPV E7-Expressing Cells

Biomolecules ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 995
Author(s):  
Xiaoyan Hou ◽  
Lijun Qiao ◽  
Ruijuan Liu ◽  
Xuechao Han ◽  
Weifang Zhang
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cell Cycle Regulation ◽  
Cell Cycle Progression ◽  
Mtor Pathway ◽  
Cycle Progression ◽  
Post Translational Modifications ◽  
Hpv E7 ◽  
Cycle Regulation

Persistent infection of high-risk human papillomavirus (HR-HPV) plays a causal role in cervical cancer. Regulator of chromosome condensation 1 (RCC1) is a critical cell cycle regulator, which undergoes a few post-translational modifications including phosphorylation. Here, we showed that serine 11 (S11) of RCC1 was phosphorylated in HPV E7-expressing cells. However, S11 phosphorylation was not up-regulated by CDK1 in E7-expressing cells; instead, the PI3K/AKT/mTOR pathway promoted S11 phosphorylation. Knockdown of AKT or inhibition of the PI3K/AKT/mTOR pathway down-regulated phosphorylation of RCC1 S11. Furthermore, S11 phosphorylation occurred throughout the cell cycle, and reached its peak during the mitosis phase. Our previous data proved that RCC1 was necessary for the G1/S cell cycle progression, and in the present study we showed that the RCC1 mutant, in which S11 was mutated to alanine (S11A) to mimic non-phosphorylation status, lost the ability to facilitate G1/S transition in E7-expressing cells. Moreover, RCC1 S11 was phosphorylated by the PI3K/AKT/mTOR pathway in HPV-positive cervical cancer SiHa and HeLa cells. We conclude that S11 of RCC1 is phosphorylated by the PI3K/AKT/mTOR pathway and phosphorylation of RCC1 S11 facilitates the abrogation of G1 checkpoint in HPV E7-expressing cells. In short, our study explores a new role of RCC1 S11 phosphorylation in cell cycle regulation.

scholarly journals EGFR-ERK induced activation of GRHL1 promotes cell cycle progression by up-regulating cell cycle related genes in lung cancer

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Yiming He ◽  
Mingxi Gan ◽  
Yanan Wang ◽  
Tong Huang ◽  
Jianbin Wang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Potential Role ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Phosphorylation Site ◽  
Promoter Regions ◽  
Cycle Progression

AbstractGrainyhead-like 1 (GRHL1) is a transcription factor involved in embryonic development. However, little is known about the biological functions of GRHL1 in cancer. In this study, we found that GRHL1 was upregulated in non-small cell lung cancer (NSCLC) and correlated with poor survival of patients. GRHL1 overexpression promoted the proliferation of NSCLC cells and knocking down GRHL1 inhibited the proliferation. RNA sequencing showed that a series of cell cycle-related genes were altered when knocking down GRHL1. We further demonstrated that GRHL1 could regulate the expression of cell cycle-related genes by binding to the promoter regions and increasing the transcription of the target genes. Besides, we also found that EGF stimulation could activate GRHL1 and promoted its nuclear translocation. We identified the key phosphorylation site at Ser76 on GRHL1 that is regulated by the EGFR-ERK axis. Taken together, these findings elucidate a new function of GRHL1 on regulating the cell cycle progression and point out the potential role of GRHL1 as a drug target in NSCLC.

Mutations at sites involved in Suc1 binding inactivate Cdc2

1991 ◽  
Vol 11 (12) ◽  
pp. 6177-6184
Author(s):  
B Ducommun ◽  
P Brambilla ◽  
G Draetta
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Specific Interaction ◽  
Physiological Role ◽  
Negative Effects ◽  
Alanine Scanning Mutagenesis ◽  
Cycle Progression ◽  
Mutant Proteins ◽  
Additional Protein

suc1+ encodes an essential cell cycle regulator of the fission yeast Schizosaccharomyces pombe. Its product, a 13-kDa protein, interacts with the Cdc2 protein kinase. Both positive and negative effects on cell cycle progression have been attributed to Suc1. To date, the exact mechanisms and the physiological role of the interaction between Suc1 and Cdc2 remain unclear. Here we have studied the molecular basis of this association. We show that Cdc2 can bind Suc1 or its mammalian homolog directly in the absence of any additional protein component. Using an alanine scanning mutagenesis method, we analyzed the interaction between Cdc2 and Suc1. We show that the integrity of several domains on the Cdc2 protein, including sites directly involved in catalytic activity, is required for binding to Suc1. Furthermore, Cdc2 mutant proteins unable to bind Suc1 (but able to bind cyclins) are nonfunctional when overexpressed in S. pombe, indicating that a specific interaction with Suc1 is required for Cdc2 function.

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