Analysis of yeast protein kinases using protein chips.
We have developed a novel protein chip technology that allows the high-throughput analysis of biochemical activities, and used this approach to analyse nearly all of the protein kinases from Saccharomyces cerevisiae. Protein chips are disposable arrays of microwells in silicone elastomer sheets placed on top of microscope slides. The high density and small size of the wells allows for high-throughput batch processing and simultaneous analysis of many individual samples. Only small amounts of protein are required. Of 122 known and predicted yeast protein kinases, 119 were overexpressed and analysed using 17 different substrates and protein chips. We found many novel activities and that a large number of protein kinases are capable of phosphorylating tyrosine. The tyrosine phosphorylating enzymes often share common amino acid residues that lie near the catalytic region. Thus, our study identified a number of novel features of protein kinases and demonstrates that protein chip technology is useful for high-throughput screening of protein biochemical activity.
Zhu
H
H
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA.
Klemic
J F
JF
Chang
S
S
Bertone
P
P
Casamayor
A
A
Klemic
K G
KG
Smith
D
D
Gerstein
M
M
Reed
M A
MA
Snyder
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D004867:Equipment Design; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020869:Gene Expression Profiling / Q000295:instrumentation* / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D010455:Peptides / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000032:analysis; D010766:Phosphorylation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D005656:Fungal Proteins; D010455:Peptides; D010761:Phosphorus Radioisotopes; D011506:Proteins; D011993:Recombinant Fusion Proteins; C416504:poly(tyrosyl-glutamic acid); D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases