Signal Transduction and Protein Phosphorylation

An intracellular signal transduction pathway, called the MAP kinase cascade, is rapidly stimulated in response to growth factors. Three enzymes in this pathway include pp90 ribosomal S6 kinase, mitogen-activated protein (MAP) kinase, and MAP kinase kinase (also referred to as MAP/ERK kinase (MEK)), which form three tiers of a protein kinase cascade in which pp90rsk is phosphorylated and activated by MAP kinase, and MAP kinase is phosphorylated and activated by MAP kinase kinase. MAP kinase kinase is itself a substrate for phosphorylation and activation by any of three protein kinases, Raf-1, MEK kinase, and Mos, and thus is a convergence point for diverse signalling pathways. The work of many laboratories has led to the definition of important connections between growth factor receptors and Raf-1. Some of these involve activation of Ras by association of the Ras-guanine nucleotide exchange factor with tyrosine kinase receptors via a linker protein, GRB2, and subsequent interaction of Ras with Raf-1 at the plasma membrane. Several cellular protooncogenes are components of this pathway. Furthermore, many transcription factors are downstream targets for MAP kinase and pp90 ribosomal S6 kinase, both of which translocate to nuclei following cell stimulation. Thus, the MAP kinase cascade is a key pathway by which external mitogenic signals control cell growth at the level of transcription and likely plays an important role in mediating oncogenic cell transformation.

Our studies are aimed at understanding the regulation of MAP kinase kinase and examining its role in tumorigenesis. We have identified several phosphorylation sites on this enzyme, and have examined their contribution to kinase activation. From this and other information, mutants of MAP kinase kinase that are either constitutively active or dominantly inactive were designed, which upon transfection into cultured cells respectively enhanced and blocked cell signal transduction through the MAP kinase pathway. One of these mutants was particularly interesting, in that internal truncation of a predicted alpha helical domain induced constitutive activation of MAP kinase kinase. Further exploration by site directed mutagenesis has led to the identification of structural components of the enzyme that are important for stabilizing its inactive conformation. We are also using the constitutively activated MAP kinase kinase mutants as tools in mammalian expression systems to identify downstream cellular targets of MAP kinase kinase and MAP kinase. We have recently demonstrated that constitutive activation of MAP kinase kinase induces mammalian cell transformation. We have also found several cell types that undergo differentiation upon overexpression of these mutants, and are characterizing the mechanisms by which these processes occur. By using several mutants ranging from high to low specific activities, it should be feasible to examine threshold effects on different downstream effectors.

Electrospray ionization mass spectrometry (ESI-MS) is a technique used in many of our studies. This is state-of-the-art technology for analyzing covalent modifications within biological molecules, and it enables the determination of protein or nucleic acid masses to accuracies of 1 in 10,000 Da. Experimental approaches we are developing in our laboratory include (i) liquid chromatography coupled to mass spectrometry (LC/MS), which separates molecules in a complex mixture by high performance liquid chromatography, followed by determination of mass, (ii) tandem mass spectrometry (MS/MS, LC/MS/MS), in which ions are selected and fragmented by collision-induced dissociation, from which information about peptide and nucleic acid sequence and specific sites of covalent modification can be obtained, and (iii) deuterium exchange coupled to mass spectrometry, which measures exchange rates from peptide backbone hydrogens, and is useful for probing higher order structure in proteins.

Using LC/MS and LC/MS/MS we have identified regulatory phosphorylation sites and autophosphorylation sites on the growth factor-regulated kinase, MAP kinase kinase, and are also examining the kinetic order of phosphorylation of its substrate, MAP kinase. We are exploring the capabilities of mass spectrometry to measure rates of deuterium exchange on MAP kinase kinase, in order to document enzyme conformational changes that result from mutagenesis. We are also using mass spectrometry technology to examine post-translational modifications of components within large protein/nucleic acid complexes, such as ribosomal 40S and 60S subunits. Of particular interest to us are novel post-translational modifications of proteins that may be under growth factor control. Currently, we are able to analyze picomole levels of peptides or proteins and are planning to extend these capabilities to the femtomole range, by applying micro techniques recently developed for ionspray mass spectrometry. By this means we aim to analyze post-translational modifications of signalling proteins derived from limited number of cells.