Magic bullets for protein kinases

Bishop, Anthony C.; Buzko, Oleksandr; Shokat, Kevan M.

doi:10.1016/s0962-8924(01)01928-6

Cited by 230 publications

(227 citation statements)

References 50 publications

Supporting

Mentioning

219

Contrasting

Unclassified

Order By: Relevance

“…2A, Right) were efficiently phosphorylated by Ypk1. GST-Orm1(1-85) was phosphorylated by an analog-sensitive mutant, Ypk1(L424A), or Ypk1-as, in the absence, but not in the presence, of the appropriate inhibitor (3-MOB-PP1) (22). By contrast, phosphorylation of the same substrate by wild-type Ypk1 was unaffected, verifying that the activity in the immunoprecipitates responsible for the observed phosphorylation was indeed Ypk1.…”

Section: Resultsmentioning

confidence: 88%

Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae

Roelants

Breslow

Muir

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

271

424

View full text Add to dashboard Cite

The Orm family proteins are conserved integral membrane proteins of the endoplasmic reticulum that are key homeostatic regulators of sphingolipid biosynthesis. Orm proteins bind to and inhibit serine: palmitoyl-coenzyme A transferase, the first enzyme in sphingolipid biosynthesis. In Saccharomyces cerevisiae, Orm1 and Orm2 are inactivated by phosphorylation in response to compromised sphingolipid synthesis (e.g., upon addition of inhibitor myriocin), thereby restoring sphingolipid production. We show here that protein kinase Ypk1, one of an essential pair of protein kinases, is responsible for this regulatory modification. Myriocin-induced hyperphosphorylation of Orm1 and Orm2 does not occur in ypk1Δ cells, and immunopurified Ypk1 phosphorylates Orm1 and Orm2 robustly in vitro exclusively on three residues that are known myriocin-induced sites. Furthermore, the temperature-sensitive growth of ypk1 ts ypk2Δ cells is substantially ameliorated by deletion of ORM genes, confirming that a primary physiological role of Ypk1-mediated phosphorylation is to negatively regulate Orm function. Ypk1 immunoprecipitated from myriocin-treated cells displays a higher specific activity for Orm phosphorylation than Ypk1 from untreated cells. To identify the mechanism underlying Ypk1 activation, we systematically tested several candidate factors and found that the target of rapamycin complex 2 (TORC2) kinase plays a key role. In agreement with prior evidence that a TORC2-dependent site in Ypk1(T662) is necessary for cells to exhibit a wild-type level of myriocin resistance, a Ypk1 (T662A) mutant displays only weak Orm phosphorylation in vivo and only weak activation in vitro in response to sphingolipid depletion. Additionally, sphingolipid depletion increases phosphorylation of Ypk1 at T662. Thus, Ypk1 is both a sensor and effector of sphingolipid level, and reduction in sphingolipids stimulates Ypk1, at least in part, via TORC2-dependent phosphorylation.cell regulation | plasma membrane

show abstract

Section: Resultsmentioning

confidence: 88%

Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae

Roelants

Breslow

Muir

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

271

424

View full text Add to dashboard Cite

show abstract

“…Mutation of L159G in PDK1 creates an enlarged ATP binding site, potentially allowing inhibition by compounds unable to bind WT kinases. This approach has been successfully applied to many protein kinases (reviewed in [21]), although it is not universally tolerated [28,29]. First we tested the activity of this mutant and its ability to be inhibited in vitro by previously described analogues of PP1, a general kinase inhibitor.…”

Section: Identification Of Inhibitor Analogues For Pdk1 Lg Inhibitionmentioning

confidence: 99%

“…Surprisingly, we noticed that the ability of BX-795 to cause a G2/M arrest was similar in PDK1 +/+ ES cells compared to PDK1 −/− ES cells, suggesting that the cell cycle consequences of this compound were unrelated to PDK1 inhibition. To achieve acute but more specific inhibition of PDK1, we employed a chemical genetic approach, whereby mutation of conserved residue(s) in its ATP binding site confers sensitivity to ATP and inhibitor analogues [21]. We mutated the large hydrophobic amino acid L159, referred to as the gatekeeper residue, to glycine (L159G, hereafter termed LG).…”

Section: Introductionmentioning

confidence: 99%

Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells

Tamgüney

Zhang

Fiedler

et al. 2008

Experimental Cell Research

View full text Add to dashboard Cite

Abstract3-Phosphoinositide-dependent kinase-1 (PDK1) phosphorylates and activates several kinases in the cAMP-dependent, cGMP-dependent and protein kinase C(AGC) family. Many putative PDK1 substrates have been identified, but have not been analyzed following transient and specific inhibition of PDK1 activity. Here, we demonstrate that a previously characterized PDK1 inhibitor, BX-795, shows biological effects that are not consistent with PDK1 inhibition. Therefore, we describe the creation and characterization of a PDK1 mutant, L159G, which can bind inhibitor analogues containing bulky groups that hinder access to the ATP binding pocket of wild type (WT) kinases. When expressed in PDK1 −/− ES cells, PDK1 L159G restored phosphorylation of PDK1 targets known to be hypophosphorylated in these cells. Screening of multiple inhibitor analogues showed that 1-NM-PP1 and 3,4-DMB-PP1 optimally inhibited the phosphorylation of PDK1 targets in PDK1 −/− ES cells expressing PDK1 L159G but not WT PDK1. These compounds confirmed previously assumed PDK1 substrates, but revealed distinct dephosphorylation kinetics. While PDK1 inhibition had little effect on cell growth, it sensitized cells to apoptotic stimuli. Furthermore, PDK1 loss abolished growth of allograft tumors. Taken together we describe a model system that allows for acute and reversible inhibition of PDK1 in cells, to probe biochemical and biological consequences.

show abstract

“…In this regard, a recent technological innovation described ATP analog-sensitive kinase alleles (ASKAs) 4 that can accommodate ATP and function normally, but that additionally have high affinity for large ATP analogs, such as 4-amino-1-tert-butyl-3-(1Ј-naphthyl)pyrazolo [3,4-d]pyrimidine (NaPP1), that compete for entry into the ATP binding site (3,4). By contrast, the active site of natural kinases is too small to accommodate such inhibitors (3) (Fig.…”

mentioning

confidence: 99%

Cutting Edge: A Chemical Genetic System for the Analysis of Kinases Regulating T Cell Development

Denzel

Hare

Zhang

et al. 2003

The Journal of Immunology

View full text Add to dashboard Cite

To understand the regulatory activities of kinases in vivo requires their study across a biologically relevant window of activity. To this end, ATP analog-sensitive kinase alleles (ASKAs) specifically sensitive to a competitive inhibitor have been developed. This article tests whether ASKA technology can be applied to complex immunological systems, such as lymphoid development. The results show that when applied to reaggregate thymic organ culture, novel p56Lck ASKAs readily expose a dose-dependent correlation of thymocyte development with a range of p56Lck activity. By regulating kinase activity, rather than amounts of RNA or protein, ASKA technology offers a general means for assessing the quantitative contributions to immunology of numerous kinases emerging from genomics analyses. It can obviate the generation of multiple lines of mice expressing different levels of kinase transgenes and should permit specific biological effects to be associated with defined biochemical activities.

show abstract

Magic bullets for protein kinases

Cited by 230 publications

References 50 publications

Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae

Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae

Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells

Cutting Edge: A Chemical Genetic System for the Analysis of Kinases Regulating T Cell Development

Contact Info

Product

Resources

About