A biochemical analysis of the MAP kinase pathway in mammalian cells

Harding, Angus Silas (2004). A biochemical analysis of the MAP kinase pathway in mammalian cells PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

       
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Author Harding, Angus Silas
Thesis Title A biochemical analysis of the MAP kinase pathway in mammalian cells
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2004
Thesis type PhD Thesis
Supervisor Prof John Hancock
Dr Brian Gabrielli
Total pages 273
Collection year 2004
Language eng
Subjects L
321020 Pathology
730108 Cancer and related disorders
Formatted abstract

The MAP kinase cascade is an evolutionarily conserved signal transduction cascade that serves multiple functions during growth and development. Cell functions regulated by the MAP kinase cascade include proliferation, differentiation, apoptosis and cell migration. One outstanding question in signal transduction is how a single linear signalling cascade is able to perform multiple biological functions. In this thesis I have performed a biochemical analysis of the MAP kinase pathway. The results of this analysis begin to address the question of how a single signalling cascade can generate multiple outputs.

Cell receptors activate the MAP kinase pathway in response to ligand stimulation by activating the small GTPase Ras. Mammals have three ubiquitously expressed Ras isoforms (H-, N-, and K-Ras) that have different biochemical activities in vivo. The amino-terminal catalytic domains (amino acids 1-165) of H-Ras, N-Ras and K-Ras are highly conserved (100% identical in residues 1-80, 95% identical in residues 80-165). All of the effector, exchange factor and nucleotide-binding sites are found in the N-terminal conserved domains. The Ras isoforms diverge in the carboxy-region, which includes the sequences that direct membrane localisation. In chapter 1 I provide direct evidence that H-Ras and K-Ras, which are targeted to the plasma membrane by different carboxy-terminal anchors, operate in functionally distinct microdomains of the plasma membrane. Different sites of plasma membrane attachment may underlie functional differences between isoforms of Ras. Palmitoylation and farnesylation targets H-Ras to lipid rafts and caveolae, but that the interaction of H-Ras with these membrane subdomains is dynamic. GTP-loading redistributes H-Ras from rafts into bulk plasma membrane by a mechanism that requires the adjacent hypervariable region of H-Ras. Release of H-Ras-GTP from rafts is necessary for efficient activation of Raf. By contrast, K-Ras is located outside rafts irrespective of bound nucleotide. These studies identify a novel protein determinant that is required for H-ras function, and show that the GTP/GDP state of H-ras determines its lateral segregation on the plasma membrane. Activation of Raf1 occurs at the plasma membrane. 14-3-3 must be complexed with Raf1 for efficient recruitment to the plasma membrane and activation by Ras, but 14-3- 3 is completely displaced from Raf1 following plasma membrane binding. I show here that interaction of Raf-1 with phosphatidylserine, an inner plasma membrane phospholipid, displaces 14-3-3 and increases Raf1 kinase activity, whereas removal of 14-3-3 from Raf-1 using specific phosphopeptides substantially reduces Raf1 basal kinase activity. Displacement of 14-3-3 from activated Rafl by phosphopeptides has no effect on kinase activity if Raf1 is first removed from solution, but completely eradicates kinase activity of soluble activated Raf1. These results suggest a mechanism for the removal of 14-3-3 from Raf-1 at the plasma membrane and show that removal of 14-3-3 from Raf-1 has markedly different effects depending on experimental conditions. In combination, these results suggest a model of Raf1 activation at the plasma membrane, where H-Ras requires access to both lipid rafts and the disordered plasma membrane in order to activate Raf1, whereas K-Ras does not.

 

In chapter 3, I report the biochemical characterisation of loss-of-function Raf mutations. Random mutagenesis and genetic screens for impaired Raf function in Caenorhabditis elegans were used to identify six loss-of-function alleles of lin-45 raf that result in a substitution of a single amino acid. The mutations were classified as weak, intermediate, and strong based on phenotypic severity. I engineered these mutations into the homologous residues of vertebrate Raf1 and analysed the mutant proteins for their underlying biochemical defects. Surprisingly, phenotype strength did not correlate with the catalytic activity of the mutant proteins. Amino acid substitutions Val-589 and Ser-619 severely compromised Raf kinase activity, yet these mutants displayed weak phenotypes in the genetic screen. Interestingly, this is because these mutant Raf proteins efficiently activate the MAPK (mitogen-activated protein kinase) cascade in living cells, a result that may inform the analysis of knockout mice. Equally intriguing was the observation that mutant proteins with non-functional Ras-binding domains, and thereby deficient in Ras-mediated membrane recruitment, displayed only intermediate strength phenotypes. This confirms that secondary mechanisms exist to couple Ras to Raf in vivo. The strongest phenotype in the genetic screens was displayed by a S508N mutation that again did not correlate with a significant loss of kinase activity or membrane recruitment by oncogenic Ras in biochemical assays. Ser-508 lies within the Raf1 activation loop, and mutation of this residue in Raf1 and the equivalent Ser-615 in B-Raf revealed that this residue regulates Raf binding to MEK. Further characterization revealed that in response to activation by epidermal growth factor, the Raf-S508N mutant protein displayed both reduced catalytic activity and aberrant activation kinetics: characteristics that may explain the C. elegans phenotype.

Activation of cyclin B-CDK1 is an absolute requirement for entry into mitosis, but other protein kinase pathways that also have mitotic functions are activated during G(2)/M progression. The MAPK cascade has well-established roles in entry and exit from mitosis in Xenopus laevis oocytes, but relatively little is known about the regulation and function of this pathway in mammalian mitosis. In chapter 4 I report a detailed analysis of the activity of all components of the Ras/Raf/MEK/ERK pathway in HeLa cells during normal G2/M. The focus of this pathway is the dramatic activation of an insoluble pool of MEK1 without the corresponding activation of the MEK substrate ERK. This is because of the uncoupling of MEK1 activation from ERK activation. The mechanism of this uncoupling involves the cyclin B-CDK1-dependent proteolytic cleavage of the N-terminal ERK-binding domain of MEK1 and the phosphorylation of Thr(286). These results demonstrate that cyclin B-CDK1 activity regulates signalling through the MAPK pathway in mitosis.

What is the function of the MAP kinase cascade during G2/M progression? MAP kinase signalling is critical for mediating spindle regulation in mammalian cells. Inhibition of MEK catalytic activity generates a significant increase in the incidence of abnormal mitoses. This likely reflects the role of ERK at kinetochores and microtubules during mitosis, as established by previous studies. In addition, MEK1 activation regulates the timing of cyclin B/CDK1 activation: activation of MEK1 during G2 delays entry into mitosis independently of the ATM/ATR checkpoint kinases. In contrast, activation of MEK1 at G2/M facilitates cyclin B/CDK1 activation. This function of MEK appears to be independent of ERK activation, raising the intriguing possibility of a novel substrate for MEK during mitosis. Strikingly, MEK1 activation during G2 and G2/M is restricted to the nucleolus. These combined results indicate that the MAP kinase pathway has multiple functions during mitosis, and that MEK1 function is determined in part by regulating its cellular localisation during G2/M progression.

A recurring theme that emerged in this thesis was that localisation is used by cells to regulate signalling output from the MAP kinase cascade. At the plasma membrane, activation of Raf1 in different microdomains generated different levels of Raf kinase activity. During mitosis, active MEK displayed highly specific localisation that correlated with distinct biological functions. It appears that localisation may be a general mechanism used by cells to generate different biological outputs from a single pathway.

Keyword Cellular signal transduction
Cellular control mechanisms
Additional Notes

*Variant title: The MAP kinase pathway in mammalian cells
*Note: Page no's 63 & 64 are repeated with different content.

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
 
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Created: Fri, 24 Aug 2007, 18:32:16 EST