Human cyclin E, originally identified on the basis of its ability to function as a G1 cyclin in budding yeast, associated with a cell cycle-regulated protein kinase in human cells. The cyclin E-associated kinase activity peaked during G1, before the appearance of cyclin A, and was diminished during exit from the cell cycle after differentiation or serum withdrawal. The major cyclin E-associated kinase in human cells was Cdk2 (cyclin-dependent kinase 2). The abundance of the cyclin E protein and the cyclin E-Cdk2 complex was maximal in G1 cells. These results provide further evidence that in all eukaryotes assembly of a cyclin-Cdk complex is an important step in the biochemical pathway that controls cell proliferation during G1.

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We have analyzed the activation of human cyclin-dependent kinases in a cell-free system. Human CDC2, cyclin-dependent kinase 2 (CDK2), cyclin A, and cyclin Bi were produced in insect cells by infection with recombinant baculoviruses. CDC2 or CDK2 monomers in lysates of infected cells could be activated by the addition of lysates containing cyclin A or Bi. CDC2 activation by cyclin B1, as well as CDK2 activation by cyclins A and Bi, was accompanied by the formation of high molecular weight complexes. In contrast, CDC2 did not bind effectively to cyclin A. CDC2 activation by cyclin Bi was studied in detail and was found to be accompanied by phosphorylation of CDC2 on Threonine 161. The binding of CDC2 to cycin Bi also occurred under conditions where CDC2 phosphorylation was prevented, resulting in an inactive complex that could then be phosphorylated and activated on addition of cell extract. Highly purified CDC2 and cyclin Bi also formed inactive complexes that could be activated in an ATP-dependent fashion by unidentified components in crude cell extracts. These data suggest that the CDC2 activation process begins with cyclin binding, after which CDC2 phosphorylation, catalyzed by a separate enzyme, leads to activation.

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Engagement of the T cell antigen receptor (TCR) by peptide antigen bound to the major histocompatibility complex (MHC) molecules initiates a biochemical cascade involving protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). Recent biochemical and genetic evidence has implicated at least three cytoplasmic protein tyrosine kinases (PTKs), Lck, Fyn, and ZAP-70, that are involved in the initiation of TCR signal transduction. In addition, genetic evidence has demonstrated the requirement of the transmembrane PTPase, CD45, for TCR function. Activation of T cells through the TCR represents an alteration in the dynamic equilibrium between PTKs and PTPases. The TCR is a multi-subunit complex composed of at least six different gene products. Dissection of the TCR utilizing chimeric receptors and TCR mutants has demonstrated that the multi-subunit receptor is composed of at least two signal transducing modules, the CD3 and the zeta chain subunits. These two modules have in common peptide sequences within their cytoplasmic domains termed antigen recognition activation motifs (ARAMs) that are responsible for transducing signaling events. Moreover, the ARAM sequence is also found in subunits associated with a variety of other hematopoietic cell antigen receptors and is likely to form the basis for interactions with effector molecules within the signaling cascades of these receptors. Here we review the mechanism by which the ARAM sequences interact with PTKs and the cascades of PTKs and PTPases that are involved in mediating TCR function.