Force production by a muscle is critical to maintaining proper function and overall health of a human or animal. Muscle adapts to increased loading with hypertrophy by activating a number of intracellular signaling cascades that regulate protein synthesis. The overall hypothesis is that force-dependent processes acutely activate growth-related signaling during active force generation. This project took two approaches. The first employed a general survey of muscles in which age-dependent changes in muscle activity differed. No conclusive activity-dependent signaling emerged however coordinated signaling among kinases broke down with age. The second approach utilized an in situ muscle preparation in which force production or metabolic costs were specifically controlled. Similar sub-maximal force levels generated by different methods found that force, per se, is not a primary modulator of growth-related signaling but that ERK phosphorylation is dependent on fiber-activation. Prolonging the duration of electrical stimulation applied to the nerve or increasing the frequency at which stimulations are applied was expected to increase the metabolic stress associated with contraction. Several growth-related kinases correlated with markers of metabolic stress, i.e. increased AMPK activity and decreased glycogen content, which were decoupled from force decline. This suggests energy depletion, specific to stimulation pattern, strongly influences the immediate response to high force contraction signaling. The overall conclusion is that signaling molecules previously implicated in force-dependent signaling lie much too downstream to relay strict force-dependent signaling.

In response to osmotic shock in Saccharomyces cerevisiae the MAP kinase Hog1 coordinates a large-scale transcriptional stress response, rapidly producing hundreds of copies of specified transcripts. Many of the most highly induced genes are bound and regulated by a transcription factor, Sko1, but lack the canonical binding site for this factor. We use ChIP-seq to demonstrate a stress-specific binding mode of Sko1. In stress, Sko1 binds to promoters in close proximity to Hog1, and another Hog1-regulated transcription factor, Hot1. This mode of Sko1 binding requires the physical presence of Hog1, but not Hog1 phosphorylation of Sko1. We identify candidate Sko1 and Hot1 binding motifs that predict co-localization of Sko1, Hot1, and Hog1 at promoters. We then demonstrate a role for Sko1 and Hot1 in directing Hog1-associated RNA Pol II to target genes, where Hog1 is present with the elongating polymerase. We suggest a possible model for Hog1 reprogramming of transcription in the early stages of the osmotic stress response. We then determine the extent and structure of the Hog1 controlled transcriptional program in a related stress, damage to the cell wall. We find that Sko1 and Hot1 have different apparent thresholds for activation by Hog1. In addition, in cell wall damage, Hog1 regulates an additional transcription factor, Rlm1, that is not involved in other Hog1 regulated stress responses. This factor is activated by the coincidence of a signal from Hog1 with that of another MAP kinase, Slt2.

The RAS/RAF/MEK/ERK pathway is the prototypical cellular signal transduction cascade and has been the focus of intense scrutiny over the last two decades. As a mitogenic pathway, its activation is a potent driver of cellular growth and survival, and its deregulation underlies many cancers. While RAS family GTPases have long been recognized as prolific human oncogenes, a landmark study in 2002 also established the RAF family kinase as a bona fide oncogene (Davies et al., 2002). Indeed, aberrant RAS-RAF signaling underlies nearly one-third of all human cancers (Wellbrock et al., 2004). Notably, mutations in RAF are found with astounding frequency in certain cancers (e.g. 70% of malignant melanomas) (Dhomen and Marais, 2007). These findings have identified intercepting aberrant RAF function as an ideal therapeutic target. RAF is a Ser/Thr protein kinase and its activity is strictly regulated by a core complex of at least three proteins, namely, KSR, CNK and HYP (Claperon and Therrien, 2007). The mechanism by which the KSR/CNK/HYP complex regulates RAF function remains enigmatic. In particular, the function of KSR in regulating RAF activity is highly controversial. The work described in this thesis was conducted with the aim of: i) understanding the interactions that underlie formation of the KSR/CNK/HYP complex, and ii) elucidating the mechanism by which the complex regulates RAF function. I have attempted to accomplish these aims using a combination of structural biology, biochemistry and cell biology approaches. I begin by presenting the structure of the SAM domain mediated interaction between CNK and HYP. I describe a model for how the CNK/HYP interaction in turn serves to recruit KSR to form the higher-order KSR/CNK/HYP complex. Subsequently, I describe the allosteric mechanism by which KSR controls RAF activation via the formation of specific side-to-side kinase domain heterodimers of KSR and RAF. Lastly, I describe a potential mechanism by which RAS directly mediates the attainment of the side-to-side dimer configuration of RAF through its own ability to form dimers. The acquisition of the side-to-side dimer configuration is essential for aberrant RAF signaling in cancers, suggesting future RAF inhibition strategies could be aimed at preventing dimer formation.

During the growth phase of oogenesis, mammalian oocytes increase several hundred-fold in volume. Although it is known that ovarian granulosa cells send growth promoting signals, neither these external signals nor the transduction pathways that become activated in the oocyte are known. Therefore, the presence and the activity of candidate signaling pathways in growing murine oocytes were investigated. By immunoblotting, the MAP kinases, ERK1 and ERK2, as well as their activating kinase MEK, were detected in oocytes at all stages of growth. However, using a phospho-specific anti-ERK antibody, no immunoreactive species were detectable in isolated granulosa cells or oocytes at any stage of growth, except metaphase II. Phosphorylated ERK was also present, although in smaller quantities, in oocyte-granulosa cell complexes at the later stages of growth. Furthermore, when ovarian sections were stained with an anti-ERK antibody, the protein was found to be highly concentrated in the cytoplasm of oocytes at all stages of growth, with lower levels in the nucleus. Another member of the MAP kinase family, Jun kinase (JNK), was investigated. By immunoblotting, JNK was detected in growing oocytes. Experiments using an anti-JNK antibody on ovary sections revealed the protein to be uniformly distributed in non-growing and growing oocytes with no evidence of preferential nuclear localization. These results imply that an interaction between the oocyte and the granulosa cells may be required to generate phosphorylated ERK. They also imply that growth signals probably are not relayed through ERK, but do not exclude a role for Jun kinase in mediating oocyte growth.

The RAS/RAF/MEK/ERK pathway is the prototypical cellular signal transduction cascade and has been the focus of intense scrutiny over the last two decades. As a mitogenic pathway, its activation is a potent driver of cellular growth and survival, and its deregulation underlies many cancers. While RAS family GTPases have long been recognized as prolific human oncogenes, a landmark study in 2002 also established the RAF family kinase as a bona fide oncogene (Davies et al., 2002). Indeed, aberrant RAS-RAF signaling underlies nearly one-third of all human cancers (Wellbrock et al., 2004). Notably, mutations in RAF are found with astounding frequency in certain cancers (e.g. 70% of malignant melanomas) (Dhomen and Marais, 2007). These findings have identified intercepting aberrant RAF function as an ideal therapeutic target. RAF is a Ser/Thr protein kinase and its activity is strictly regulated by a core complex of at least three proteins, namely, KSR, CNK and HYP (Claperon and Therrien, 2007). The mechanism by which the KSR/CNK/HYP complex regulates RAF function remains enigmatic. In particular, the function of KSR in regulating RAF activity is highly controversial. The work described in this thesis was conducted with the aim of: i) understanding the interactions that underlie formation of the KSR/CNK/HYP complex, and ii) elucidating the mechanism by which the complex regulates RAF function. I have attempted to accomplish these aims using a combination of structural biology, biochemistry and cell biology approaches. I begin by presenting the structure of the SAM domain mediated interaction between CNK and HYP. I describe a model for how the CNK/HYP interaction in turn serves to recruit KSR to form the higher-order KSR/CNK/HYP complex. Subsequently, I describe the allosteric mechanism by which KSR controls RAF activation via the formation of specific side-to-side kinase domain heterodimers of KSR and RAF. Lastly, I describe a potential mechanism by which RAS directly mediates the attainment of the side-to-side dimer configuration of RAF through its own ability to form dimers. The acquisition of the side-to-side dimer configuration is essential for aberrant RAF signaling in cancers, suggesting future RAF inhibition strategies could be aimed at preventing dimer formation.

In C. albicans, a MAP kinase pathway has been implicated in aspects of controlling hyphal development. We have examined the transcription profile of cells deleted for the transcription factor Cph1 as well as Cst20, Hst7 and Cek1, three upstream kinases potentially involved in Cph1 regulation. Deletion of these four elements does not block filament induction by serum and does not dramatically affect the transcription profile of yeast-hyphal transition, but deletion of CPH1 delays filamentation. Over-expression of Cph1 by ADH1pt-CPH1 significantly enhances filamentation, suggesting that Cph1 is helpful but not essential for filament induction. Interestingly, the transcription profile of ADH1pt-CPH1 expressing cells under yeast conditions is similar to that of wild type strains undergoing the yeast-hyphal transition. Finally, it appears that Cek1 and its regulators Hst7 and Cst20 may control the repression of genes such as CHT2 through a process independent of the Cph1p transcription factor.

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Pharmacology. Includes bibliographical references.

Diss. (sammanfattning) Umeå : Umeå Universitet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.