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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...

1995; Nesterov et al., 1995a;Ohno et al., 1995;Heilker et al., 1996). The α chain of the AP-2 heterotetramer can Many plasma membrane proteins destined for endobind clathrin (Goodman and Keen, 1995), synaptotagmin cytosis are concentrated into clathrin-coated pits (Zhang et al., 1994), Eps15 (Benmerah et al., 1995 Tebar through the recognition of a tyrosine-based motif ), Grb2 (Okabayashi et al., 1996 and small their cytosolic domains by an adaptor (AP-2) complex.phosphorylated molecules like inositol phosphates and The μ2 subunit of isolated AP-2 complexes binds phosphoinositides (Beck and Keen, 1991a; Timerman specifically, but rather weakly, to proteins bearing et al., 1992;Voglmaier et al., 1992; Gaidarov et al., 1996). the tyrosine-based signal. We now demonstrate, usingThe physiological significance of these interactions is peptides with a photoreactive probe, that this binding under investigation. The β chain contacts clathrin and is strengthened significantly when the AP-2 complex drives coat assembly (Ahle and Ungewickell, 1989; is present in clathrin coats, indicating that there is Schroder and Ungewickell, 1991; Gallusser and cooperativity between receptor-AP-2 interactions and Shih et al., 1995). The μ2 chain coat formation. Phosphoinositides with a phosphate at recognizes the tyrosine-based endocytic motif (Ohno et al., the D-3 position of the inositol ring, but not other 1995; Boll et al., 1996), a sequence of four amino isomers, also increase the affinity of the AP-2 complex acids of the form tyrosine-polar-polar-large hydrophobic for the tyrosine-based motif. AP-2 is the first protein (YppØ), which is used for sorting proteins from the plasma known (in any context) to interact with phosphatidylmembrane to the endosome (Trowbridge et al., 1993; inositol 3-phosphate. Our findings indicate that recep- Thomas and Roth, 1994). tor recruitment can be coupled to clathrin coatThe interactions just described do not by themselves assembly and suggest a mechanism for regulation of explain how cargo recruitment and coat formation are membrane traffic by lipid products of phosphoinositide coupled and how concentration of cargo into coated 3-kinases.structures is achieved. Previous work has shown that the Keywords: adaptors/clathrin/coated pits/membrane isolated AP-2 complex, or even its isolated μ2 subunit, traffic/protein sorting can recognize the tyrosine-based motif (Ohno et al., 1995;Boll et al., 1996). This interaction reflects the specificity seen in vivo, but it is rather weak (dissociation constant Introduction~1 0 μM). Are there regulatory mechanisms that enhance

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Phosphoinositide (PI) 3-kinase and the mitogen-activated protein (MAP) kinase cascades are activated by many of the same ligands. Several groups have reported involvement of PI 3-kinase in the activation of Erk1 and Erk2, whereas many other groups have shown that activation of Erk1 and Erk2 is not sensitive to inhibitors of PI 3-kinase such as wortmannin. Here we show that wortmannin inhibition of the MAP kinase pathway is cell type-and ligand-specific. Wortmannin blocks platelet-derived growth factor (PDGF)-dependent activation of Raf-1 and the MAP kinase cascade in Chinese hamster ovary cells, which have few PDGF receptors, but has no significant effect on Erk activation in Swiss 3T3 cells, which have high levels of PDGF receptors. However, wortmannin blocks activation of Erk proteins if Swiss 3T3 cells are stimulated with lower, physiological levels of PDGF. These results suggest that PI 3-kinase is in an efficient pathway for activation of MAP kinase, but that MAP kinase can be stimulated by a redundant pathway when a large number of receptors are activated. We present evidence that a protein kinase C family member downstream of phospholipase C␥ is involved in the redundant pathway. PI1 3-kinase has been implicated as being involved in the signal transduction of virtually all growth factors studied and in the transformation of cells by several oncoproteins (1, 2). Activation of PI 3-kinase with growth factors results in the appearance of the lipid products of this enzyme, PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 , within seconds to minutes. There is also a correlation between the elevation in PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 levels and cell transformation. These correlations have suggested an important role for PI 3-kinase in signal transduction pathways leading to cell growth and transformation. Indeed, the catalytic subunit of PI 3-kinase (p110␣) has recently been identified as a retroviral oncogene (3). Overexpression of p110␣ in chick embryo fibroblasts results in constitutive elevation of PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 and cell transformation.The MAP kinase pathway is another key component in the transduction of signals leading to growth and transformation. This pathway consists of a linear cascade of the protein kinases Raf, MEK, and MAP kinase/Erk; like PI 3-kinase, Erk1 and Erk2 are acutely activated by growth factors and are found constitutively activated in many transformed cell lines. The Erk proteins are phosphorylated and activated by the dual specificity kinase MEK (MAP kinase/Erk kinase), which is phosphorylated and activated by the serine/threonine kinase Raf. Raf is recruited to the membrane of activated cells by direct binding to Ras-GTP. This recruitment by activated Ras is necessary but not sufficient for full activation of Raf (4); therefore, other factors that are necessary for activation of the MAP kinase pathway may feed into the pathway at Raf. A search for other sources that feed into the MAP kinase pathway has yielded several candidates including Src, members of the PKC family, ...

Homodimerization of the erythropoietin (EPO) receptor (EPO-R) in response to EPO binding transiently activates the receptor-associated protein tyrosine kinase JAK2. Tyrosine phosphorylation of the EPO-R creates ''docking sites'' for SH2 domain(s) in signaling molecules such as the protein tyrosine phosphatases SH-PTP1 and SH-PTP2, phosphoinositide 3-kinase (PI3 kinase), and STAT5. However, little is known about the specific intracellular signals essential for proliferation and differentiation of erythroid progenitors. Here we show that an EPO-R containing only one cytosolic (phospho)tyrosine residue, Y479, induces a signal transduction pathway sufficient for proliferation and differentiation of fetal liver progenitors of erythroid colony-forming units from EPO-R ؊͞؊ mice as well as for proliferation of cultured hematopoietic cells. This cascade involves sequential EPOinduced recruitment of PI3 kinase to the EPO-R and activation of mitogen-activated protein kinase activity, independent of the Shc͞Grb2-adapter pathway and of STAT5. Protein kinase C may be one of the mediators connecting PI3 kinase with the mitogen-activated protein kinase signaling cascade. Our results identify a signaling cascade important in vivo for erythroid cell proliferation and differentiation.Erythropoietin (EPO), a hormone synthesized by the kidney, is the primary regulator of mammalian erythropoiesis. Signaling through the EPO receptor (EPO-R) (1), which is a member of the cytokine receptor family, regulates proliferation, differentiation and survival of erythroid progenitor cells. Homodimerization of the receptor in response to EPO binding (2, 3) transiently activates the receptor-associated protein tyrosine kinase JAK2 (4). Tyrosine phosphorylation of the EPO-R creates ''docking sites'' for SH2 domain(s) in signaling molecules such as the protein tyrosine phosphatases SH-PTP1 (5) and SH-PTP-2 (6), phosphoinositide 3-kinase (PI3 kinase) (7), and STAT5 (8-10). However, little is known about the specific intracellular signals essential for proliferation and differentiation of erythroid progenitors.In a number of growth factor receptor systems recruitment of PI3 kinase correlates with mitogenic signaling (11). This lipid kinase is a heterodimeric enzyme composed of the regulatory p85 subunit and the catalytic p110 subunit that phosphorylates phosphoinositides at the D3 position of the inositol ring. Stimulation by many cytokines including EPO (7, 12, 13) results in a transient increase in PI3 kinase activity. Mediated by the SH2-domains of p85, recruitment of PI3 kinase to specific phosphotyrosine residues within the cytoplasmic domain of receptors places the enzyme in close proximity to substrates. Recent evidence suggests that the lipid products act as second messengers and directly activate enzymes such as Akt (protein kinase B) (14) and certain isoforms of protein kinase C (PKC) (15, 16). Proposed downstream targets of PI3 kinase include p70 S6 kinase and MEK [mitogen-activated protein (MAP) kinase (MAPK) kinase]. The latter wo...