No abstract

Phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2), a key molecule in the phosphoinositide signalling pathway, was thought to be synthesized exclusively by phosphorylation of PtdIns-4-P at the D-5 position of the inositol ring. The enzymes that produce PtdIns-4,5-P2 in vitro fall into two related subfamilies (type I and type II PtdInsP-5-OH kinases, or PIP(5)Ks) based on their enzymatic properties and sequence similarities'. Here we have reinvestigated the substrate specificities of these enzymes. As expected, the type I enzyme phosphorylates PtdIns-4-P at the D-5 position of the inositol ring. Surprisingly, the type II enzyme, which is abundant in some tissues, phosphorylates PtdIns-5-P at the D-4 position, and thus should be considered as a 4-OH kinase, or PIP(4)K. The earlier error in characterizing the activity of the type II enzyme is due to the presence of contaminating PtdIns-5-P in commercial preparations of PtdIns-4-P. Although PtdIns-5-P was previously thought not to exist in vivo, we find evidence for the presence of this lipid in mammalian fibroblasts, establishing a new pathway for PtdIns-4,5-P2 synthesis.

Xenopus oocytes, which are arrested in G2 of meiosis I, contain complexes of cyclin B-cdc2 (M phase-promoting factor) that are kept repressed by inhibitory phosphorylations on cdc2 at Thr-14 and Tyr-15. Progesterone induces a cytoplasmic signaling pathway that leads to activation of cdc25, the phosphatase that removes these phosphorylations, catalyzing entry into M phase. It has been known for 25 years that high levels of cAMP and protein kinase A (PKA) are required to maintain the G 2 arrest and that a drop in PKA activity is required for M phase-promoting factor activation, but no physiological targets of PKA have been identified. We present evidence that cdc25 is a critical target of PKA. (i) In vitro, cdc25 Ser-287 serves as a major site of phosphorylation by PKA, resulting in sequestration by 14-3-3. (ii) Endogenous cdc25 is phosphorylated on Ser-287 in oocytes and dephosphorylated in response to progesterone just before cdc2 dephosphorylation and M-phase entry. (iii) High PKA activity maintains phosphorylation of Ser-287 in vivo, whereas inhibition of PKA by its heat-stable inhibitor (PKI) induces dephosphorylation of Ser-287. (iv) Overexpression of mutant cdc25 (S287A) bypasses the ability of PKA to maintain oocytes in G 2 arrest. These findings argue that cdc25 is a physiologically relevant target of PKA in oocytes. In the early embryonic cell cycles, Ser-287 is phosphorylated during interphase and dephosphorylated just before cdc2 activation and mitotic entry. Thus, in addition to its role in checkpoint arrest, cdc25 Ser-287 serves as a site for regulation during normal, unperturbed cell cycles.

The T-cell antigen CD28 provides a costimulatory signal that is required for T-cefl proliferation. (residues 191-194). Mutation of the Y191 within the motif resulted in a complete loss ofbinding, while mutation ofM194 caused partial loss of binding. Bding analysis showed that the CD28 Y(P)-MXM motif bound to the p85 C-and N-terminal SH2 domans with an afnity comparable to that observed for PDGF-R and insulin receptor substrate 1. In terms of snaling, CD28 ligation induced a dramatic increase in the recruitment and association of PI 3-kinase with the receptor. CD28 is likely to use PI 3-klipase as the second signal leading to T-cell proliferation, an event with implications for anergy and peripheral T-cell tolerance.Activation of the resting T lymphocyte involves at least two steps: an antigen-specific signal generated by the T-cell receptor (TcR)2/CD3 and CD4/CD8-p56lck complexes, followed by a second signal delivered by an accessory cell (1). The CD28 antigen predominates in providing the costimulatory signals for T-cell stimulation (ref. 2; for reviews see refs. 1 and 3). It contains a single immunoglobulin-like domain and a 51-amino acid cytoplasmic tail (4). B7-1 and related B7-2/ B70 are also members of the immunoglobulin supergene family and serve as ligands for CD28 (5-7). Suboptimal proliferation induced by antigen, anti-CD3 or anti-CD2 antibody is augmented by CD28 ligation (for review see ref. 8). Likewise, CD28 ligation by B7 serves as an obligatory second step in T-cell activation (for review see ref.3). CD28-transgenic mice exhibit profound defects in mitogenic responses (9). CD28 signaling stabilizes mRNA for various lymphokines, in particular interleukin 2 (reviewed in refs. 1 and 8). T-cell nonresponsiveness or anergy can be reversed by CD28 engagement (1-3, 10), an event of potential importance in the generation of cytolytic responses against tumors (11,12).An important question concerns the mechanism by which CD28 regulates T-cell growth and the manner in which the CD28 signaling pathway relates to signals generated by the TcRC/CD3-CD4 complex. CD4 and CD8 associate with the protein-tyrosine kinase p56lck, while TcRC/CD3 is cou- By contrast, intracellular events generated by CD28 differ from those induced by TcRC/CD3 ligation. CD28 signaling appears to occur independent of conventional phosphatidylinositol hydrolysis and the Ca2 -dependent cyclophilincalcineurin pathway and is resistant to cyclosporin and FK506 (3,8). CD28 signaling, however, is sensitive to inhibitors of tyrosine kinases and to coligation with the protein-tyrosine phosphatase CD45 (3,8). CD28 ligation in the presence of phorbol ester induced the tyrosine phosphorylation of proteins at 75 and 100 kDa (3,8). This potentially unique pathway leads to DNA binding activity at an element located in the 5' interleukin 2 enhancer region (for review see refs. 1 and 3). PI 3-kinase is a heterodimer consisting of an adaptor subunit (p85) with two SH2 domains that is coupled to a p110 catalytic subunit and is important to signali...