Regulation of cytokine-independent survival kinase (CISK) by the Phox homology domain and phosphoinositides (original) (raw)
Related papers
The Journal of biological chemistry, 2001
Phosphoinositide 3-kinases (PI3Ks) are dual specificity lipid and protein kinases. While the lipid-dependent PI3K downstream signaling is well characterized, little is known about PI3K protein kinase signaling and structural determinants of lipid substrate specificity across the various PI3K classes. Here we show that sequences C-terminal to the PI3K ATP-binding site determine the lipid substrate specificity of the class IA PI3K␣ (p85/ p110␣). Transfer of such activation loop sequences from class II PI3Ks, class III PI3Ks, and a related mammalian target of rapamycin (FRAP) into p110␣ turns the lipid substrate specificity of the resulting hybrid protein into that of the donor protein, while leaving the protein kinase activity unaffected. All resulting hybrids lacked the ability to produce phosphatidylinositol 3,4,5trisphosphate in intact cells. Amino acid substitutions and structure modeling showed that two conserved positively charged (Lys and Arg) residues in the activation loop are crucial for the functionality of class I PI3Ks as phosphatidylinositol 4,5-bisphosphate kinases. By transient transfecion of 293 cells, we show that p110␣ hybrids, although unable to support lipid-dependent PI3K signaling, such as activation of protein kinase B/Akt and p70 S6k , retain the capability to associate with and phosphorylate insulin receptor substrate-1, with the same specificity and higher efficacy than wild type PI3K␣. Our data lay the basis for the understanding of the class I PI3K substrate selectivity and for the use of PI3K␣ hybrids to dissect PI3K␣ function as lipid and protein kinase.
Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation
The Biochemical journal, 1998
While a plethora of extracellular molecules exist that modulate cellular functions via binding to membrane receptors inside the cell, their actions are mediated by relatively few signalling mechanisms. One of these is activation of phosphatidylinositol 3-kinase (PI-3K), which results in the generation of a membrane-restricted second messenger, polyphosphatidylinositides containing a 3'-phosphate. How these molecules transduced the effects of agonists of PI-3K was unclear until the recent discovery that several protein kinases become activated upon exposure to 3'-phosphorylated inositol lipids. These enzymes include protein kinase B (PKB)/AKT and PtdIns(3,4, 5)P3-dependent kinases 1 and 2, the first two of which interact with 3'-phosphorylated phosphoinositides via pleckstrin homology domains. Once targeted to the membrane by this motif, PKB becomes phosphorylated at two residues, which relieves intermolecular inhibition, allowing the activated complex to dissociate and m...
Molecular and cellular biology, 1997
Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors after growth factor stimulation of cells. Activation of PI 3-kinase results in increased intracellular levels of 3' phosphorylated inositol phospholipids and the induction of signaling responses, including the activation of the protein kinase Akt, which is also known as RAC-PK or PKB. We tested the possibility that the phospholipid products of PI 3-kinase directly mediate the activation of Akt. We have previously described a constitutively active PI 3-kinase, p110, which can stimulate Akt activity. We used purified p110 protein to generate a series of 3' phosphorylated inositol phospholipids and tested whether any of these lipids could activate Akt in vitro. Phospholipid vesicles containing PI3,4 bisphosphate (P2) specifically activated Akt in vitro. By contrast, the presence of phospholipid vesicles containing PI3P or PI3,4,5P3 failed to increase the...
PKB/Akt: Functional Insights from Genetic Models
Nature Reviews Molecular Cell Biology, 2001
Recent characterization of PKB/AKT and its upstream regulators-such as phosphoinositide-dependent kinase 1 (PDK1)-as important signal transducers of phosphatidylinositol 3-kinase (PI3K)-derived signalling has uncovered essential roles for these and related serine/threonine kinases in the control of transcription and protein translation, which impact on cell growth, survival and metabolism. This review discusses some of the most recent studies examining PKB/AKT regulation and function, as well as highlighting the emerging evidence that similar kinases, such as the serum and glucocorticoid activated kinase (SGK), might actually perform some of the roles originally attributed to PKB/AKT. History and significance of PKB/AKT PKB/AKT was first thrust into the limelight with the finding that it is activated by 3′ phospholipids-the products of class I PI3Ks (BOX 1). Several cellular stimulants that act through receptor tyrosine kinases or Gprotein-coupled receptors lead to enhanced PI3K activity and elevated levels of its products, phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P 2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P 3). These lipids show an affinity for certain protein modules, such as the PLECKSTRIN HOMOLOGY (PH) DOMAIN. As the 3′ phosphatidylinositol phospholipid concentrations rise, PH-domain-containing proteins are recruited to the membrane. Franke and co-workers first showed lipid dependence of PKB/AKT activation through PH-domain interaction with the products of PI3K 1,2. In the case of PKB/AKT, this translocation brings it into proximity with another PH-domain-containing protein kinase, PDK1, resulting in PKB/AKT phosphorylation (discussed in more detail below). This generates an active form of the protein, which targets specific proteins in the cytoplasm and nucleus (FIG. 1). PKB/AKT is the cellular homologue of the transforming oncogene of the AKT8 oncovirus (v-Akt) 3. In METAZOANS, there are three PKB/AKT genes, which yield PKBα/AKT1, PKBβ/AKT2 and PKBγ/ΑKT3 (FIG. 2). All three gene products are similar in structure and size, contain an amino-terminal PH domain and seem to be regulated by similar mechanisms (described below), albeit with some distinctions 4. The PKBs fall into the AGC (protein kinase A (PKA)/protein kinase G/protein kinase C-like) class of protein kinases and, like other members of this class, require phosphorylation for activation. With the exception of a spliced isoform of PKBγ/AKT3, this involves phosphorylation at two residues: a threonine (residue 308 in PKBα/AKT1) within the protein kinase 'T-LOOP' and a serine buried in a hydrophobic motif proximal to the carboxyl terminus (serine 473 in PKBα/AKT1) (REF. 5). Mutation of these residues to alanine severely reduces activation, whereas substitution with aspartic acid generates a PI3K-independent, constitutively active enzyme. Activation of PKB/AKT PKB/AKT phosphorylation at Thr308. A common characteristic shared with the AGC kinase family is the requirement for phosphorylation of a flexible peptide loop (T-loop) proximal to the catalytic core of the kinase
Negative regulation of phosphatidylinositol 3-kinase and Akt signalling pathway by PKC
Cellular Signalling, 2003
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2) is an important second messenger in signaling pathways in organisms ranging from yeast to mammals, but the regulation of PI(4,5)P 2 levels remains unclear. Here we present evidence that PI(4,5)P 2 levels in Saccharomyces cerevisiae are down-regulated by the homologous and functionally redundant proteins TAX4 and IRS4. The EPS15 homology domain-containing proteins TAX4 and IRS4 bind and activate the PI(4,5)P 2 5-phosphatase INP51 via an Asn-Pro-Phe motif in INP51. Furthermore, the INP51-TAX4/IRS4 complex negatively regulates the cell integrity pathway. Thus, TAX4 and IRS4 are novel regulators of PI(4,5)P 2 and PI(4,5)P 2-dependent signaling. The interaction between TAX4/IRS4 and INP51 is analogous to the association of EPS15 with the 5-phosphatase synaptojanin 1 in mammalian cells, suggesting that EPS15 is an activator of synaptojanin 1.
The EMBO Journal, 1999
Phosphoinositide 3-kinases (PI3Ks) are lipid kinases which also possess an in vitro protein kinase activity towards themselves or their adaptor proteins. The physiological relevance of these phosphorylations is unclear at present. Here, the protein kinase activity of the tyrosine kinase-linked PI3K, p110δ, is characterized and its functional impact assessed. In vitro autophosphorylation of p110δ completely down-regulates its lipid kinase activity. The single site of autophosphorylation was mapped to Ser1039 at the C-terminus of p110δ. Antisera specific for phospho-Ser1039 revealed a very low level of phosphorylation of this residue in cell lines. However, p110δ that is recruited to activated receptors (such as CD28 in T cells) shows a timedependent increase in Ser1039 phosphorylation and a concomitant decrease in associated lipid kinase activity. Treatment of cells with okadaic acid, an inhibitor of Ser/Thr phosphatases, also dramatically increases the level of Ser1039-phosphorylated p110δ. LY294002 and wortmannin blocked these in vivo increases in Ser1039 phosphorylation, consistent with the notion that PI3Ks, and possibly p110δ itself, are involved in the in vivo phosphorylation of p110δ. In summary, we show that PI3Ks are subject to regulatory phosphorylations in vivo similar to those identified under in vitro conditions, identifying a new level of control of these signalling molecules.
The Biochemical journal, 2014
The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates PtdIns (phosphatidylinositol) at endosomal membranes to generate PtdIns(3)P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing PtdIns(3)P-binding PX (phox homology) and FYVE domains. In the present study, we describe a highly selective and potent inhibitor of Vps34, termed VPS34-IN1, that inhibits Vps34 with 25 nM IC50 in vitro, but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific PtdIns(3)P-binding probe from endosome membranes, within 1 min, without affecting the ability of class I PI3K to regulate Akt. Moreover, we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3), the only protein kinase known to interact specific...
Identification of CISK, a new member of the SGK kinase family that promotes IL3-dependent survival
Current Biology, 2000
The signaling pathways for cell survival are much less well understood than those for apoptosis [1]. Many mammalian cell-survival factors have been identified, either biochemically or from genetic studies in other organisms. Effective genetic methods that allow systematic study of anti-apoptosis genes in mammalian cells remain to be established, however. To achieve this goal, we used a new genetic screening method using enhanced retroviral mutagen (ERM) vectors to identify factors that mediate IL-3-dependent survival of hematopoietic cells. Both known and novel mediators of cell survival were identified, including Bcl-xL, phosphatidylinositide 3-kinase (PI 3-kinase), Akt and cytokine-independent survival kinase (CISK). CISK is a PX-domain-containing serine/threonine kinase homologous to serum- and glucocorticoid-regulated protein kinase (SGK). We showed that CISK acts downstream of the PI 3-kinase cascade in vivo and may function in parallel to Akt by phosphorylating Bad and the transcription factor FKHRL1. The distinct subcellular localization of CISK, however, suggests that it acts in different signaling cascades from Akt. Our results demonstrate the power of ERM to identify key genes involved in cell-survival signaling. Furthermore, CISK is the first SGK family member that has been shown to promote survival, pointing to the possibility that other SGK family proteins may also function in survival pathways.