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Cyclin-dependent kinases (Cdks) are principal drivers of cell division and are an important therapeutic target to inhibit aberrant proliferation. Cdk enzymatic activity is tightly controlled through cyclin interactions, posttranslational modifications, and binding of inhibitors such as the p27 tumor suppressor protein. Spy1/RINGO (Spy1) proteins bind and activate Cdk but are resistant to canonical regulatory mechanisms that establish cell-cycle checkpoints. Cancer cells exploit Spy1 to stimulate proliferation through inappropriate activation of Cdks, yet the mechanism is unknown. We have determined crystal structures of the Cdk2-Spy1 and p27-Cdk2-Spy1 complexes that reveal how Spy1 activates Cdk. We find that Spy1 confers structural changes to Cdk2 that obviate the requirement of Cdk activation loop phosphorylation. Spy1 lacks the cyclin-binding site that mediates p27 and substrate affinity, explaining why Cdk-Spy1 is poorly inhibited by p27 and lacks specificity for substrates with cyclin-docking sites. We identify mutations in Spy1 that ablate its ability to activate Cdk2 and to proliferate cells. Our structural description of Spy1 provides important mechanistic insights that may be utilized for targeting upregulated Spy1 in cancer.

Cardiac ATP-sensitive [K.sup.+] ([K.sub.ATP]) channels are formed by Kir6.2 and SUR2A subunits. We produced transgenic mice that express dominant negative Kir6.x pore-forming subunits (Kir6.1-AAA or Kir6.2-AAA) in cardiac myocytes by driving their expression with the [alpha]-myosin heavy chain promoter. Weight gain and development after birth of these mice were similar to nontransgenic mice, but an increased mortality was noted after the age of 4-5 mo. Transgenic mice lacked cardiac [K.sub.ATP] channel activity as assessed with patch clamp techniques. Consistent with a decreased current density observed at positive voltages, the action potential duration was increased in these mice. Some myocytes developed EADs after isoproterenol treatment. Hemodynamic measurements revealed no significant effects on ventricular function (apart from a slightly elevated heart rate), whereas in vivo electrophysiological recordings revealed a prolonged ventricular effective refractory period in transgenic mice. The transgenic mice tolerated stress less well as evident from treadmill stress tests. The proarrhythmogenic features and lack of adaptation to a stress response in transgenic mice suggest that these features are intrinsic to the myocardium and that [K.sub.ATP] channels in the myocardium have an important role in protecting the heart from lethal arrhythmias and adaptation to stress situations. potassium channels; ATP-sensitive [K.sup.+] channel; heart; ventricle; stress responses doi:10.1152/ajpheart.00051.2006

The decision for a cell to self-replicate requires passage from G1 to S phase of the cell cycle and initiation of another round of DNA replication. This commitment is a critical one that is tightly regulated by many parallel pathways. Significantly, these pathways converge to result in activation of the cyclin-dependent kinase, cdk2. It is, therefore, important to understand all the mechanisms regulating cdk2 to determine the molecular basis of cell progression. Here we report the identification and characterization of a novel cell cycle gene, designated Speedy (Spy1). Spy1 is 40% homologous to the Xenopus cell cycle gene, X-Spy1. Similar to its Xenopus counterpart, human Speedy is able to induce oocyte maturation, suggesting similar biological characteristics. Spy1 mRNA is expressed in several human tissues and immortalized cell lines and is only expressed during the G1/S phase of the cell cycle. Overexpression of Spy1 protein demonstrates that Spy1 is nuclear and results in enhanced cell proliferation. In addition, flow cytometry profiles of these cells demonstrate a reduction in G1 population. Changes in cell cycle regulation can be attributed to the ability of Spy1 to bind to and prematurely activate cdk2 independent of cyclin binding. We demonstrate that Spy1-enhanced cell proliferation is dependent on cdk2 activation. Furthermore, abrogation of Spy1 expression, through the use of siRNA, demonstrates that Spy1 is an essential component of cell proliferation pathways. Hence, human Speedy is a novel cell cycle protein capable of promoting cell proliferation through the premature activation of cdk2 at the G1/S phase transition.

A study was conducted on the role of QLS motif in transmembrane helix VII of the glucose transporter family. The findings indicate that the QLS motif in helix VII functions as a molecular filter that dictates the ability of GLUTs 1, 3 and 4 to transport glucose. Moreover, this region of helix VII plays a key role in substrate recognition at the exofacial substrate binding site by interaction with the C-1 position of the incoming sugar molecule.

Alanine-scanning mutagenesis of putative transmembrane helix VIII and an investigation of the role of proline residues in transport catalysis were conducted to understand the structure-function of the brain-type glucose transporter, GLUT3. Results reveal that no individual amino acid within the helix is required for transport catalysis. Results also suggest that the role of the helix is to provide a framework for structural packing within the transporter molecule.

A range of chimeric glucose transporters composed of regions of liver-type (GLUT2) and brain-type (GLUT3) human facilitative glucose transporters were produced to determine the regions of the transporter which are involved in substrate recognition and binding. Findings indicate a model in which the seventh putative transmembrane-spanning helix is intimately involved in the selection of transported substrate and in which this region plays a critical role in determining the K(sub m) for 2-deoxyglucose.