Wes Yonemoto

Prokaryotic expression of catalytic subunit of adenosine cyclic monophosphate-dependent protein kinase

The prokaryotic expression of the Cα subunit of cAPK provides a system for the production of milligram quantities of wild-type and mutant protein with relative ease and little expense. The protocols described here have been optimized for the production of soluble and active protein kinase, with kinetic parameters similar to those found for the enzyme isolated from mammalian tissue. The Cα subunit expressed in E. coli is a phosphoprotein and appears to contain the sites of phosphorylation identified in the mammalian protein. The free N-terminal Gly indicates the lack of an N-terminal myristic acid; however, coexpression of the gene encoding the yeast N-myristoyltransferase allows for myristoylated Cα subunit to be produced in E. coli. Finally, the purified recombinant Cα subunit has been used in X-ray crystallographic studies which have yielded the crystals that will allow the first three-dimensional structure of a protein kinase to be solved. Therefore, although the employment of prokaryotic expression in the production of functional protein kinases has been limited, it is hoped that the methods presented here for the C subunit of cAPK will encourage the use of this simple and versatile expression system.

Mammalian cAMP-dependent protein kinase functionally replaces its homolog in yeast

The cDNA encoding the catalytic subunit (C alpha) from mouse cAMP-dependent protein kinase (PK) was expressed in Saccharomyces cerevisiae. By a plasmid swap procedure, we demonstrated that the mammalian C alpha subunit can functionally replace its yeast homolog to maintain the viability of a yeast strain containing genetic disruptions of the three TPK genes encoding the yeast C subunits. C alpha subunit produced in yeast was purified and its biochemical properties were determined. The protein isolated from yeast appears to be myristylated, as has been found for C subunits from higher eukaryotic cells. This system would be useful for studying the biochemistry of the mammalian enzyme in vitro and its biological role in a model in vivo system. These studies demonstrate that the PK substrate(s) required for viability are recognized by the mammalian enzyme. In general terms, these results demonstrate that heterologous proteins with only 50% sequence conservation with their yeast counterparts can be functional in yeast. This is an important result because it validates the use of yeast to identify the biological role of newly cloned genes from heterologous systems, a key tenet of the Human Genome Initiative.

The Catalytic Subunit of cAMP-Dependent Protein Kinase

The protein kinase catalytic core in essence comprises an extended network of interactions that link distal parts of the molecule to the active site where they facilitate phosphoryl transfer from ATP to protein substrate. This review defines key sequence and structural elements, describes what is currently known about the molecular interactions, and how they are involved in catalysis.

[51] Functional expression of mammalian adenosine cyclic monophosphate-dependent protein kinase in saccharomyces cerevisiae

The heterologous expression of protein kinases in E. coli has proved difficult and unpredictable. Although the v-abl protein kinase is successfully expressed in E. coli, our experiments on expression of yeast C subunits in E. coli produced large amounts of predominantly insoluble and inactive protein. Attempts to refold the protein proved unsuccessful. In contrast, a major fraction of mouse C alpha expressed in E. coli is soluble and the enzyme in the soluble fraction is active; however, certain mutant forms have proved to be unstable, difficult to purify, or insoluble. In addition, the E. coli system cannot be used to study the biological role of posttranslational modifications specific to eukaryotic systems. Several protein kinases have been expressed in soluble form in insect cells using baculovirus, suggesting that this system is generally more reliable than E. coli. However, the presence and nature of posttranslational modifications in insect cells may be different from that found in the natural source and may affect the biochemical function. In addition, baculovirus expression is not particularly useful for studying biological questions. Mouse C alpha and C beta have been overexpressed in NIH3T3 cells. This approach is useful in characterizing the biochemical properties of C alpha versus C beta, but it may not be an ideal system for studying mutant proteins since wild-type C subunits are still expressed from the chromosomal copies in this genetic background. This small level of wild type may make it difficult to analyze weakly functional mutants, which have activities less than 10% that of wild type. Several cell lines with altered subunits of cAMP-dependent protein kinase have been identified but a strain completely devoid of C subunit has not been adequately characterized for protein structure/function studies. Disruption of the genes encoding cAMP-dependent protein kinase in mammalian cells has not yet been accomplished. This chapter describes a method to express a C subunit of mammalian cAMP-dependent kinase in yeast. We have demonstrated that the mouse C alpha subunit can substitute for its yeast counterpart. Since at least one functional C subunit is required for viability, these results suggest that the yeast substrates important for viability are recognized by the mammalian C subunit. Although the sequence conservation between yeast and mouse C subunit is only about 50%, these results demonstrate that heterologous proteins with relatively low sequence conservation with their yeast counterparts can be functional in yeast.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein Kinase Structure & Function: cAMP-Dependent Protein Kinase

Protein phosphorylation is a major mechanism for regulation in eukaryotic cells, and the protein kinases represent a large and very diverse family of enzymes. Nearly all major metabolic pathways are regulated at some step by phosphorylation. In addition, protein kinases play critical roles in mitogenesis, in cell cycle events, and in many types of oncogenesis. One of the first protein kinases to be discovered was cAMP-dependent protein kinase (cAPK) (Walsh DA, et al., 1968). In the intervening decades, the family has grown to well over 100. These enzymes are complex and differ in terms of their size, subunit structure, subcellular localization, and mechanism of activation. cAPK, however, remains as one of the simplest. Furthermore, despite the diversity of the kinases, all share a conserved catalytic core that is included within the free catalytic (C) subunit of cAPK (Hanks SK, et al., 1988). Thus, the C-subunit can serve as a framework for the entire family (Figure 1).