Cdc25B is a phosphatase that catalyzes the dephosphorylation and activation of the cyclin-dependent kinases, thus driving cell cycle progression. We have identified two residues, R488 and Y497, located >20 Å from the active site, that mediate protein substrate recognition without affecting activity toward small-molecule substrates. Injection of Cdc25B wild-type but not the R488L or Y497A variants induces germinal vesicle breakdown and cyclin-dependent kinase activation in Xenopus oocytes. The conditional knockout of the cdc25 homolog (mih1) in Saccharomyces cerevisiae can be complemented by the wild type but not by the hot spot variants, indicating that protein substrate recognition by the Cdc25 phosphatases is an essential and evolutionarily conserved feature. protein-protein interactions The hot spot theory of protein-protein interactions suggests that the binding energy between two proteins is governed in large part by just a few critical residues at the binding interface. In typical interfaces of 1,200-2,000 Å 2 , only Ϸ5% of the residues from each protein contribute Ͼ2 kcal͞mol to the binding interaction (1). Although the establishment of the hot spot theory in pioneering studies by the Wells and Fersht laboratories using the high-affinity systems of human growth hormone binding protein (2) and barnase-barstar (3) has been subsequently validated in other similar systems, it is less clear to what extent hot spot theory applies to the transient protein-protein interactions involved in enzyme catalysis. Given that many such transient interactions govern crucial intracellular signaling processes (for example, protein kinases and phosphatases), it is of great interest to identify and characterize such potential hot spots. Identification of these hot spots would allow for experiments that probe the details of specific transient protein-protein interactions in vivo. Additionally, such sites could serve as targets for the development of small-molecule compounds that interfere specifically with these protein-protein interactions and serve as leads for drug development.Cdc25 phosphatases are regulators of the eukaryotic cell cycle that dephosphorylate and thus activate the main gatekeepers of this process, the cyclin-dependent kinases (Cdks͞cyclins) (4). Confirming their importance in cell cycle regulation, Cdc25A and Cdc25B contribute to oncogenic transformation and are overexpressed in many diverse types of cancer (5). Crystal structures of the catalytic domains of Cdc25A and Cdc25B have revealed a shallow active site with no obvious features for mediating substrate recognition, suggesting a broad protein interface rather than a lock-and-key interaction (6, 7). This finding is confirmed by the activity of the Cdc25 phosphatases toward Cdk͞cyclin protein substrates, which is six orders of magnitude greater than that of peptidic substrates that contain the same primary sequence (8). The shallow active site also correlates with the lack of potent and specific inhibitors of the Cdc25 phosphatases, despite extensive eff...

The length of the G1 phase in the cell cycle shows significant variability in different cell types and tissue types. To gain insights into the control of G1 length, we generated an E2F activity reporter that captures free E2F activity after dissociation from Rb sequestration and followed its kinetics of activation at the single-cell level, in real time. Our results demonstrate that its activity is precisely coordinated with S phase progression. Quantitative analysis indicates that there is a pre-S phase delay between E2F transcriptional dynamic and activity dynamics. This delay is variable among different cell types and is strongly modulated by the cyclin D/CDK4/6 complex activity through Rb phosphorylation. Our findings suggest that the main function of this complex is to regulate the appropriate timing of G1 length.

The Rb/E2F pathway plays a central role in regulating cell-fate decisions and cell-cycle progression. The E2F1 protein, a major effector of the pathway, is regulated via a combination of transcriptional, translational and posttranslational constraints. Elucidating the regulation and impact of the Rb/E2F pathway requires direct measurement of E2F1 dynamics in single cells.To this end, we have engineered fluorescent E2F1 protein reporters to enable live detection and quantification in single cells. The reporter constructs expressed an E2F1-Venus fusion protein under the regulation of the mouse or human E2F1 promoter and contained or excluded the 3'UTR of the E2F1 gene, a sequence that contains miRNA regulatory regions that modulate expression of the protein. Expression of the reporter protein was highly dynamic during the cell cycle: there was no or little fluorescent signal in G 0 , but levels steadily increased during late G 1 and peaked during mid to late S phase before returning to baseline before the onset of mitosis.The absence of the E2F1 3'UTR in the constructs led to considerably higher steady-state levels of the fusion protein, which although normally regulated, exhibited a slightly less complex dynamic profile during the cell cycle or genotoxic stress. Lastly, the presence or absence of Rb failed to impact in substantial ways the overall detection and levels of the reporters.

Problem Most medical schools lack parental leave policies, leaving medical students vulnerable to discrimination, diminished educational opportunities, delays in graduation or matching, and breaches of privacy. This report outlines the steps taken by student-leaders to advocate for such a policy and the lessons learned along the way. Approach In September 2018, leaders of the Family Support Initiative, a medical student interest group at the University of North Carolina School of Medicine, initiated the process of advocating for a clear, official parental leave policy. Certain elements proved essential in bringing about institutional change, including active involvement of a faculty advocate; well-documented student testimonials; commitment from top administrative leaders; involvement of the Title IX office and legal counsel; creating space for authentic collaboration; building clear, flexible mechanisms for making up missed time; and consideration of preclinical training and regional campuses. Outcomes The Education Committee unanimously approved the New Child Adjustment Policy in June 2019. The policy was published online; shared broadly in various formats with students, faculty, and members of the university health system; and announced at class meetings and new student orientations. Faculty advisors were trained on its content and procedures. Administrators and students report that the policy has informed their discussions around family planning and made these conversations easier to navigate. Next Steps The process model outlined here is intended to serve as a roadmap for other institutions. While student input should inform the development of parental leave policies, institutions are morally and ethically responsible for providing parental leave policies that address the key components outlined here. The authors will further study the impact of this policy on student satisfaction and academic performance. The authors urge the Liaison Committee on Medical Education and Commission on Osteopathic College Accreditation to make clear, inclusive, student-centered parental leave policies a requirement for accreditation.

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