The cyclin/cyclin-dependent kinase (Cdk) complexes and the Cdk inhibitors (CDKI) are crucial regulators of cell cycle progression in all eukaryotic cells. Using rat cardiac myocytes as a model system, this chapter provides a detailed account of methods that can be employed to measure both c ...

The Wee kinases (Wee1, Wee2, and Myt1) are major regulators of mitotic entry. They function by phosphorylating Cdc2 and related Cdks on conserved tyrosine and threonine residues. This phosphorylation blocks the activity of the Cdc2 and prevents entry into mitosis. The abundance and activi ...

Most cyclin-dependent kinases are negatively regulated by phosphorylation of two residues, a threonine at residue 14 and a tyrosine at residue 15. These residues are dephosphorylated by the cdc25 family of dual-specificity phosphatases leading to cell cycle progression. These pho ...

Polo-like kinase-1 (Plk-1) is an important cell cycle regulatory kinase that has been implicated in a multitude of cell cycle events. In this chapter we review those multiple functions of Plk-1 and describe the methods routinely used in our laboratory to purify Plk-1 from cellular lysates and mea ...

Eukaryotic cells regulate progression through the cell cycle in response to DNA damage. Cell cycle checkpoints are the signal transduction pathways that couple the detection of DNA damage to the proteins that control transitions in the cell cycle. The protein kinase Chk1, originally dis ...

The Ipl1/Aurora family of protein kinases are required for accurate chromosome segregation. Because members of this family are often overexpressed in human tumors, they have recently received much attention, both from the academic community and the pharmaceutical industry. Inde ...

The formation of specific protein—protein interactions is one of the key mechanisms for signal transduction mediated by tyrosme phosphorylation. These intermolecular interactions target signaling proteins to particular cellular locations and modulate the enzymatic ac ...

The past decade has seen an explosion in our understanding of the mechamsms underlying the transmission of signals from outside the cell, and the ways in which those signals are interpreted and integrated within the cell. This progress comcides with an appreciation that regulated, stable pr ...

The binding of extrinsic proteins to membranes is important for their biological activity in many different systems. Consider a few examples from the calcium/phospholipid second-messenger system (reviewed in refs. 1 and 2). First, membrane binding facilitates interaction of the Gq p ...

The coordmation of cell growth, differentiation, and other activities in a multicellular organism is precisely controlled by a plethora of growth factors or cytokines that achieve then effects upon the cell by binding to specific cell-surface receptors. The majority of these numerous r ...

Farnesyl-protein transferase (FPTase) catalyzes the postiranslational addition of the IScarbon isoprenoid, farnesyl, to approx 20 cellular proteins. Farnesylation is essential for the membrane localization and function of the modified proteins. Among these proteins are t ...

Rho-family signal-transduction cascades, which appear to be conserved in all eukaryotes, probably coordinate cytoskeletal reorganization and transcriptional activation via a variety of protein and lipid kinases, and other as-yet-undefined target proteins. The dissecti ...

Dual-speclflcity phosphatases, a subfamily enzymes of protein tyrosine phosphatases, play important roles in signal transduction and cell-cycle regulation during cell growth and differentiation (1). Unlike classic tyrosme phosphatases which are specific for phosphoty ...

Rho-related GTPase proteins, Rat 1,2, Rho A, B, G, and CDC42Hs, constitute a distinct subfamily in the Ras super family of GTPases (1) During the last few years a large body of evidence has been accumulated that suggests that Rho-like proteins play a critical role in the organization of the actin cytoskele ...

The substrate specificity of a protein tyrosine kinase in viva reflects both the intrinsic specificity of the kinase catalytic domain and the effective local concentrations of protein substrates. In many cases, the distribution of potential substrates is mfluenced by interactions ...

Recent inqui-res into the molecular mechanisms underlying cellular behavior have underscored the importance of dynamic associations between proteins, or protein-protein interactions, in regulating a variety of brological processes. These protein-protein interacti ...

The molecular identification and characterization of the components of receptor-signaling pathways has revealed a striking redundancy and diversity of signaling elements. For example, G protein-coupled receptors bind to a diversity of ligands, ranging from classical low-mo ...

Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) act as transducers and amplifiers between activated heptahelical membrane receptors and effector systems such as enzymes, ion channels, and transporters to mediate signals from the outside to insi ...

The oocytes of several organisms—most frequently those of the African clawed toad Xenopus laevis—have been used for many years as an excellent system to study regulation of transcription, translation, protein modification processes, secretion, and protein compartmentalizat ...

Microinjection has been widely used as a technique to introduce proteins and DNA into mammalian cells. A major advantage of mrcroinjection over transfection approaches IS that tt is possible to analyze very early responses to proteins; responses to microinjected proteins can be detect ...