Heng Zhu

Global analysis of protein phosphorylation in yeast

Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology, the in vitro substrates recognized by most yeast protein kinases: we identified over 4,000 phosphorylation events involving 1,325 different proteins. These substrates represent a broad spectrum of different biochemical functions and cellular roles. Distinct sets of substrates were recognized by each protein kinase, including closely related kinases of the protein kinase A family and four cyclin-dependent kinases that vary only in their cyclin subunits. Although many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, indicating possible new roles for several kinases. Furthermore, integration of the phosphorylation results with protein–protein interaction and transcription factor binding data revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast. Because many yeast proteins and pathways are conserved, these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.

Protein chip technology

Microarray technology has become a crucial tool for large-scale and high-throughput biology. It allows fast, easy and parallel detection of thousands of addressable elements in a single experiment. In the past few years, protein microarray technology has shown its great potential in basic research, diagnostics and drug discovery. It has been applied to analyse antibody-antigen, protein-protein, protein-nucleic-acid, protein-lipid and protein-small-molecule interactions, as well as enzyme-substrate interactions. Recent progress in the field of protein chips includes surface chemistry, capture molecule attachment, protein labeling and detection methods, high-throughput protein/antibody production, and applications to analyse entire proteomes.

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.

Protein analysis on a proteomic scale

The long-term challenge of proteomics is enormous: to define the identities, quantities, structures and functions of complete complements of proteins, and to characterize how these properties vary in different cellular contexts. One critical step in tackling this goal is the generation of sets of clones that express a representative of each protein of a proteome in a useful format, followed by the analysis of these sets on a genome-wide basis. Such studies enable genetic, biochemical and cell biological technologies to be applied on a systematic level, leading to the assignment of biochemical activities, the construction of protein arrays, the identification of interactions, and the localization of proteins within cellular compartments.

'Omic' approaches for unraveling signaling networks

Signaling pathways are crucial for cell differentiation and response to cellular environments. Recently, a large number of approaches for the global analysis of genes and proteins have been described. These have provided important new insights into the components of different pathways and the molecular and cellular responses of these pathways. This review covers genomic and proteomic (collectively referred to as omic) approaches for the global analysis of cell signaling, including gene expression profiling and analysis, protein-protein interaction methods, protein microarrays, mass spectroscopy and gene-disruption and engineering approaches.

Workshop I – Global Analysis of Protein Activities Using Protein Chips

The genomes of a wide variety of organisms have now been sequenced; a major challenge ahead is to understand the function, regulation and modification of the many encoded gene products. We have been carrying out proteomics approaches to the identification and analysis of signalling pathways in yeast. 121 of 122 protein kinases were cloned and purifed from yeast as GST fusions and analyzed for their ability to phosphorylate 60 different yeast substrates. More than 93% of the kinases exhibited activities that are 5 fold or higher, relative to controls, including 18 of 24 previously uncharacterized kinases. Many protein kinases had novel activities; for example 27 yeast kinases were found to phosphorylate Tyr. In addition, we have now cloned 6000 open reading frames and overexpressed their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with a variety of different proteins, nucleic acids and phospholipids. As examples, we have probed yeast proteome chips with calmodulin and six different phospholipids. These studies revealed many new calmodulin and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. They can also be used to screen protein-drug interactions and to detect posttranslational modifications.