Design and Use of Analog‐Sensitive Protein Kinases

Justin Blethrow1, Chao Zhang1, Kevan M. Shokat1, Eric L. Weiss2

1 University of California at San Francisco, San Francisco, California, 2 Northwestern University, Evanston, Illinois
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.11
DOI:  10.1002/0471142727.mb1811s66
Online Posting Date:  May, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Many protein kinases can be engineered to accept analogs of ATP that are not efficiently used by wild‐type kinases. These engineered kinases, which are referred to as “analog‐sensitive” or “–as” alleles, are also often sensitive to protein kinase inhibitor variants that do not block the activity of nonmutant kinases. Selective in vitro use of radiolabeled ATP analogs by –as kinases can be exploited to identify the direct phosphorylation targets of individual kinases in complex extracts. In organisms in which it is practical to replace wild‐type kinase genes with engineered alleles, the in vivo activity of a –as kinase can be reversibly blocked with an allele‐specific inhibitor. Thus, analog‐sensitive kinases can be effective tools for discovery of the cellular functions and phosphorylation targets of individual enzymes. A theoretical background for the design and use of these alleles is discussed, as are strategies for construction of candidate –as alleles of any kinase.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Synthesis of 1NM‐PP1
  • Basic Protocol 2: Synthesis of 1NA‐PP1
  • Basic Protocol 3: Preparation of γ‐32P‐Labeled N6(Benzyl)ATP
  • Support Protocol 1: Preparation of Cobalt Affinity Resin (IDA‐Co2+‐Sepharose)
  • Support Protocol 2: Expression and Purification of NDPK
  • Basic Protocol 4: In Vivo Inhibition of Analog‐Sensitive Kinases in Yeast
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Synthesis of 1NM‐PP1

  Materials
  • 1‐naphthylacetic acid (Acros Organics)
  • Hexane
  • N,N‐dimethylformamide (DMF; Aldrich)
  • Oxalyl chloride (Aldrich)
  • Tetrahydrofuran (THF; Aldrich)
  • Sodium hydride (Aldrich)
  • Malononitrile (Aldrich)
  • 1 N H 2SO 4
  • Ethyl acetate
  • MgSO 4, anhydrous
  • 1,4‐dioxane (Aldrich)
  • Sodium bicarbonate (Aldrich)
  • Dimethyl sulfate (Aldrich)
  • Diethyl ether (Et 2O)
  • Silica Gel 60 (pore size 0.040 to 0.063 mm; Merck)
  • Ethanol (Aldrich)
  • Triethylamine (Aldrich)
  • tert‐butylhydrazine hydrochloride (Aldrich)
  • Chloroform (CHCl 3)
  • 1% (v/v) methanol in CHCl 3
  • Formamide (Aldrich)
  • Activated charcoal
  • Celite
  • Büchi Rotavapor Model R‐200 or equivalent rotary evaporator
  • Ice bath
  • Separatory funnel
  • Oil baths, 80°, 100°C, 180°C
  • 10‐in. (25.4‐cm) length × 2‐in. (5.0‐cm) i.d. chromatography column
  • TLC plates (Silica Gel F 254; EM Science) and tank
  • Reflux condenser
  • Filter paper
  • Buchner funnel

Basic Protocol 2: Synthesis of 1NA‐PP1

  Materials
  • 1‐naphthoyl chloride (Aldrich)
  • Tetrahydrofuran (THF; Aldrich)
  • Sodium hydride (Aldrich)
  • Malononitrile (Aldrich)
  • 1 N H 2SO 4
  • Ethyl acetate
  • MgSO 4, anhydrous
  • 1,4‐dioxane (Aldrich)
  • Sodium bicarbonate (Aldrich)
  • Dimethyl sulfate (Aldrich)
  • Diethyl ether (Et 2O)
  • Silica Gel 60 (pore size 0.040 to 0.063 mm; Merck)
  • Sodium ethoxide (Acros Organics)
  • tert‐butylhydrazine hydrochloride (Aldrich)
  • N,N‐dimethylformamide (DMF; Aldrich)
  • Chloroform (CHCl 3)
  • 1% (v/v) methanol in CHCl 3
  • Formamide (Aldrich)
  • Ethanol (Aldrich)
  • Activated charcoal
  • Celite
  • Separatory funnel
  • Oil baths, 80°, 100°, and 180°C
  • 10‐in. (25.4‐cm) length × 2‐in. (5.0‐cm) i.d. and 8‐in. (20.3‐cm) length × 1.5‐in. (3.8‐cm) i.d. chromatography columns
  • TLC plates (Silica Gel 60 F 254; EM Science) and tank
  • Reflux condenser
  • Filter paper
  • Buchner funnel

Basic Protocol 3: Preparation of γ‐32P‐Labeled N6(Benzyl)ATP

  Materials
  • 1:1 slurry of cobalt affinity resin (IDA‐Co2+‐Sepharose) in HBS/0.02% sodium azide (see protocol 4)
  • HEPES buffered saline (HBS): 150 mM NaCl/100 mM HEPES, pH 7.4
  • Purified NDPK‐6×His (see protocol 5)
  • Phosphate‐buffered saline (PBS): 150 M NaCl/100 mM sodium phosphate pH 7.4 (see appendix 22 for sodium phosphate buffer)
  • [γ‐32P]ATP (∼3000 Ci/mmol or ∼7000 Ci/mmol)
  • 1 mM N6(benzyl)ADP (Shah et al., ; Shah and Shokat, )
  • PBS (see above) containing 5 mM MgCl 2
  • 2‐mm glass beads (VWR)
  • 1‐ml disposable pipet tip
  • Stand and clamp to accommodate 1‐ml disposable pipet tip
NOTE: All steps are performed at room temperature.

Support Protocol 1: Preparation of Cobalt Affinity Resin (IDA‐Co2+‐Sepharose)

  Materials
  • Iminodiacetic acid (IDA)–Sepharose slurry (Sigma)
  • 200 mM cobalt chloride
  • HEPES‐buffered saline (HBS): 150 mM NaCl/100 mM HEPES, pH 7.4
  • HBS (see above) containing 0.02% sodium azide
  • 15‐ml conical tubes

Support Protocol 2: Expression and Purification of NDPK

  Materials
  • pJDB1 expression plasmid: available from the Shokat Laboratory ( ) or Weiss Laboratory ( )
  • E. coli strain BL21(DE3) (Novagen)
  • IPTG
  • Cobalt affinity resin (IDA‐Co2+‐Sepharose; see protocol 4)
  • HIK200 buffer (see recipe), 4°C
  • HEK10 buffer (see recipe), 4°C
  • HKG buffer (see recipe), 4°C
  • Liquid nitrogen
  • 30°C shaking incubator
  • 5‐ml chromatography column
  • MWCO 15,000 dialysis membrane
  • Additional reagents and equipment for transformation of E. coli (unit 1.8), growing bacterial cultures (unit 1.2), preparation of bacterial lysates (unit 1.7), dialysis ( appendix 3C), and determination of protein concentration (unit 10.1)

Basic Protocol 4: In Vivo Inhibition of Analog‐Sensitive Kinases in Yeast

  Materials
  • Inhibitors: INM‐PP1 (see protocol 1) and INA‐PPI (see protocol 2)
  • DMSO
  • Yeast cells of interest containing wild‐type (control) and analog‐sensitive kinase alleles
  • Appropriate liquid and solid yeast media (unit 13.1)
  • 5‐mm sterile filter discs
  • Additional reagents and equipment for growing yeast cells (unit 13.2)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 18.11.1 Synthetic schemes of 1NM‐PP1 and 1NA‐PP1. Conditions: (i) 5 eq oxalyl chloride, 0.1 eq DMF, room temperature, 1 hr; (ii) 2 eq NaH, 1 eq malononitrile, THF, 0°C to room temperature, 1 hr; (iii) 8 eq NaHCO3, 5 eq dimethyl sulfate, dioxane/water (6:1), reflux, 1 hr; (iv) 2 eq triethylamine, 1 eq tert‐butylhydrazine hydrochloride, EtOH, reflux, 1 hr; (v) 1 eq tert‐butylhydrazine, DMF, room temperature, 1 hr; (vi) formamide, 180°C, 10 hr.
  •   FigureFigure 18.11.2 NMR data for 1‐ tert‐butyl‐3‐naphthalen‐1ylmethyl‐1 H‐pyrazolo[3,4‐ d]pyrimidin‐4‐ylamine (1NM‐PP1). Compound is white powder; 1H NMR (CDCl3, 400 Mhz) δ 1.82 (s, 9H), 4.73 (s, 2H), 4.87 (br s, 2H), 7.17 (d, J = 7 Hz, 1H), 7.37 (t, J = 8 Hz, 1H), 7.52 (m, 2H), 7.78 (d, J = 8 Hz, 1H), 7.87 (m, 1H), 8.19 (m, 1H), 8.23 (s, 1H); 13C NMR (CDCl3, 100 Mhz) δ 29.2, 32.7, 60.0, 101.1, 123.5, 125.6, 125.8, 126.2, 126.6, 128.2, 128.9, 131.9, 133.9, 134.0, 140.5, 154.5, 154.7, 157.6, HRMS (EI) molecular ion calculated for C20H21N5 331.17993, found 331.17951.
  •   FigureFigure 18.11.3 NMR data for 1‐ tert‐butyl‐3‐naphthalen‐1‐yl‐1 H‐pyrazolo[3,4‐ d]pyrimidin‐4‐ylamine (1NA‐PP1). Compound is white powder; 1H NMR (CDCl3, 400 Mhz) δ 1.87 (s, 9H), 5.04 (br s, 2H), 7.05 (m, 2H), 7.58 (t, J = 8 Hz, 1H), 7.64 (d, J = 7 Hz, 1H), 7.92 (m, 2H), 7.95 (d, J = 8 Hz, 1H), 8.36 (s, 1H); 13C NMR (CDCl3, 100 Mhz) δ 29.3, 60.6, 101.5, 125.5, 125.6, 126.5, 127.1, 128.4, 128.4, 129.6, 130.6, 131.8, 134.0, 140.4, 153.9, 154.7, 157.7, HRMS (EI) molecular ion calculated for C19H19N5 317.16427, found 317.16247.
  •   FigureFigure 18.11.4 Inhibitor binding. Important contacts between PP1 and the ATP binding site of the Src‐family protein kinase Hck (left) and the space created in the – as1 allele that allows 1NA‐PP1 binding (right). The right panel is inferred from the structure of c‐Src(‐ as1).
  •   FigureFigure 18.11.5 Subdomain V and VII alignments. Analog‐sensitive alleles of these protein kinases, whose wild‐type sequences ar shown, have been engineered. The gatekeeper residue in each kinase and the site of the second mutation (immediately amino terminal to the DFG motif) in Cla4 and JNK are shaded in gray.
  •   FigureFigure 18.11.6 Hypothetical data from – as kinase inhibition experiments. (A) Inhibition curves for a kinase‐dependent metabolic process in which the amount of metabolite consumed in a fixed time can be measured. (B) Simple morphometric analysis of the inhibition of a kinase required for normal cell morphogenesis. Percent of normal wild‐type cells is omitted for clarity.

Videos

Literature Cited

Literature Cited
   Bishop, A.C., Shah, K., Liu, Y., Witucki, L., Kung, C., and Shokat, K.M. 1998. Design of allele‐specific inhibitors to probe protein kinase signaling. Curr. Biol. 8:257‐266.
   Bishop, A., Kung, C., Shah, K., Witucki, L., Shokat, K.M., and Liu, Y. 1999. Generation of monospecific nanomolar tyrosine kinase inhibitors via a chemical genetic approach. J. Am. Chem. Soc. 121:627‐631.
   Bishop, A.C., Ubersax, J.A., Petsch, D.T., Matheos, D.P., Gray, N.S., Blethrow, J., Shimizu, E., Tsien, J.Z., Schultz, P.G., Rose, M.D., Wood, J.L., Morgan, D.O., and Shokat, K.M. 2000. A chemical switch for inhibitor‐sensitive alleles of any protein kinase. Nature 407:395‐401.
   Bishop, A.C., Buzko, O., and Shokat, K.M. 2001. Magic bullets for protein kinases. Trends Cell Biol. 11:167‐72.
   Carroll, A.S., Bishop, A.C., DeRisi, J.L., Shokat, K.M., and O'Shea, E.K. 2001. Chemical inhibition of the Pho85 cyclin‐dependent kinase reveals a role in the environmental stress response. Proc. Nat. Acad. Sci. U.S.A. 98:12578‐12583.
   Druker, B.J., Sawyers, C.L., Capdeville, R., Ford, J.M., Baccarani, M., and Goldman, J.M. 2001. Chronic myelogenous leukemia. Hematology (Am. Soc. Hematol. Educ. Program) pp. 87‐112.
   Gray, N.S., Wodicka, L., Thunnissen, A.M., Norman, T.C., Kwon, S., Espinoza, F.H., Morgan, D.O., Barnes, G., LeClerc, S., Meijer, L., Kim, S.H., Lockhart, D.J., and Schultz, P.G. 1998. Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors. Science 281:533‐538.
   Hanks, S.K. and Hunter, T. 1995. Protein kinases 6. The eukaryotic protein kinase superfamily: Kinase (catalytic) domain structure and classification. FASEB J. 9:576‐596.
   Kraybill, B.C., Elkin, L.L., Blethrow, J.D., Morgan, D.O., and Shokat, K.M. 2002. Inhibitor scaffolds as new allele specific kinase substrates. J. Am. Chem. Soc. 124:12118‐12128.
   Mourad, N. and Parks, R.E. Jr. 1966. Erythrocytic nucleoside diphosphokinase. II. Isolation and kinetics. J. Biol. Chem. 241:271‐278.
   Polson, A.G., Huang, L., Lukac, D.M., Blethrow, J.D., Morgan, D.O., Burlingame, A.L., and Ganem, D. 2001. Kaposi's sarcoma‐associated herpesvirus K‐bZIP protein is phosphorylated by cyclin‐dependent kinases. J. Virol. 75:3175‐3184.
   Schindler, T., Sicheri, F., Pico, A., Gazit, A., Levitzki, A., and Kuriyan, J. 1999. Crystal structure of Hck in complex with a Src family‐selective tyrosine kinase inhibitor. Mol. Cell 3:639‐648.
   Sekiya‐Kawasaki, M., Groen, A.C., Cope, M.J., Kaksonen, M., Watsonn, H.A., Zhang, C., Shokat, K.M., Wendland, B., McDonald, K.L., McCaffery, J.M., and Drubin, D.G. 2003. Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real‐time chemical genetic analysis. J. Cell Biol. 162:765‐772.
   Shah, K. and Shokat, K.M. 2002. A chemical genetic screen for direct v‐Src substrates reveals ordered assembly of a retrograde signaling pathway. Chem. Biol. 9:35‐47.
   Shah, K., Liu, Y., Deirmengian, C., and Shokat, K.M. 1997. Engineering unnatural nucleotide specificity for Rous sarcoma virus tyrosine kinase to uniquely label its direct substrates. Proc. Natl. Acad. Sci. U.S.A. 94:3565‐3570.
   Ubersax, J.A., Woodbury, E.L., Quang P.N., Paraz, M., Blethrow, J.D., Shah, K., Shokat, K.M., and Morgan, D.O. 2003. Targets of the cyclin‐dependent kinase Cdk1. Nature 425:859‐864.
   Wang, H., Shimizu, E., Tang, Y.P., Cho, M., Kyin, M., Zuo, W., Robinson, D.A., Alaimo, P.J., Zhang, C., Morimoto, H., Zhuo, M., Feng, R., Shokat, K.M., and Tsien, J.Z. 2003. Inducible protein knockout reveals temporal requirement of CaMKII reactivation for memory consolidation in the brain. Proc. Natl. Acad. Sci. U.S.A. 100:4287‐4292.
   Weaver, R.H. 1962. Nucleoside diphosphokinases. Enzymes 6:151‐160.
   Weiss, E.L., Bishop, A.C., Shokat, K.M., and Drubin, D.G. 2000. Chemical genetic analysis of the budding‐yeast p21‐activated kinase Cla4p. Nat. Cell Biol. 2:677‐685.
   Weiss, E.L., Kurischko, C., Zhang, C., Shokat, K., Drubin, D.G., and Luca, F.C. 2002. The Saccharomyces cerevisiae Mob2p‐Cbk1p kinase complex promotes polarized growth and acts with the mitotic exit network to facilitate daughter cell‐specific localization of Ace2p transcription factor. J. Cell Biol. 158:885‐900.
   Witucki, L.A., Huang, X., Shah, K., Liu, Y., Kyin, S., Eck, M.J., and Shokat, K.M. 2002. Mutant tyrosine kinases with unnatural nucleotide specificity retain the structure and phospho‐acceptor specificity of the wild‐type enzyme. Chem. Biol. 9:25‐33.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library