The Raf-1 serine/threonine protein kinase plays a central role in many of the mitogenic signaling pathways regulating cell growth and differentiation. The regulation of Raf-1 is complex, and involves protein-protein interactions as well as changes in the phosphorylation state of Raf-1 that are accompanied by alterations in its electrophoretic mobility. We have previously shown that a 33-kDa COOH-terminal, kinase-inactive fragment of Raf-1 underwent a mobility shift in response to the stimulation of cells with serum or phorbol esters. Here we demonstrate that treatment of NIH 3T3 cells or Sf9 cells with hydrogen peroxide (H 2 O 2 ) also induces the mobility shift of the kinase-inactive Raf-1 fragment. A series of deletion mutants of the Raf-1 COOH terminus were analyzed, and the region required for the mobility shift was localized to a 78-amino acid fragment (residues 566 -643). Metabolic labeling revealed that the slower migrating forms of the 33-kDa and of the smaller fragment contained phosphorus. Mutation of a previously characterized phosphorylation site, serine 621, to alanine prevented the mobility shift as well as phosphate incorporation or Src and Ras-dependent kinase activation in Sf9 cells when this mutation was engineered into the full-length Raf-1. Mutation of 621 to aspartate yielded a protein that existed in both the shifted and unshifted forms, demonstrating that a negative charge at 621 was necessary, but not sufficient, for the mobility shift to occur; however, its full-length form was still resistant to activation in the Sf9 system. Additional mutation of nearby serine 624 to alanine blocked the shift, implicating this residue as the site of the second of a two-step modification process leading to the slower migrating form. Co-expression of the 33-kDa fragment with an activated form of mitogen-activated protein kinase kinase in NIH 3T3 led to the appearance of the shifted form in a serum-independent manner. These results demonstrate that a mitogen-activated protein kinase kinase-induced event involving modification of serines 621 and 624 leads to the mobility shift of Raf-1.Raf-1, the product of the c-raf-1 protooncogene, is a cytoplasmic serine/threonine protein kinase that is activated in response to various mitogenic signals (1, 2). Activation of Raf-1 results in a cascade of events involving the phosphorylation and subsequent activation of additional proteins, including mitogen-activated protein (MAP) 1 kinase kinase (MEK) and MAP kinase, which ultimately lead to transcriptional activation and mitogenesis (3, 4). Thus the Raf-1 protein kinase plays a central role in cellular signal transduction and is a key player in transmitting signals from the plasma membrane to the nucleus (5, 6).The amino terminus of Raf-1 serves as a regulatory domain, while the catalytic domain resides in the carboxyl terminus, which also may contain regulatory elements (7). Earlier studies showed that the activation of Raf-1 by membrane-associated, protein-tyrosine kinases was dependent on cellular Ras activity (8 -...

We have previously shown that vimentin is a growth-regulated gene whose mRNA levels increase after serum stimulation of quiescent hamster fibroblasts. In this study, the control of the growth-regulated expression of vimentin was determined in ts13 cells induced to proliferate by serum. Both transcriptional and post-transcriptional mechanisms of regulation were examined by determining transcriptional rates, cytoplasmic transcript abundance, transcript stability, and protein abundance. We observed a fourfold increase in vimentin transcripts in the cytoplasm of serum-stimulated ts13 cells. Since transcripts are stable in both quiescent and stimulated cells, this induction of vimentin expression is a result of a fivefold increase in vimentin-specific transcriptional activity. As a result of this increased transcript availability, the abundance of polymerized vimentin protein increased following serum stimulation of quiescent fibroblasts. Overall, the induction of vimentin expression in fibroblasts by serum is a consequence of increased vimentin-specific transcriptional activity. The significance of this with regard to cytoskeletal organization and cell division is discussed.

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This study analyzed how teaching assistant(s) (TA) training affects TA attitudes and student achievement during infusion of inquiry into an introductory biology lab. A respiration lab was redesigned to incorporate a more inquiry based approach. TA attitudes towards the new lab, TA confidence in students' understanding of material, and student achievement were monitored for two years. During year one (Y1) of the study, TA's (n=18) were trained on the mechanics of the lab, but received minimal instruction on how to teach in an inquiry based fashion. The second year (Y2) of the study, TA's (n=15) received extensive training on the benefits of inquiry based education and training on the mechanics of the lab. Survey results indicate that when educated on the benefits of inquiry based education, TA's were more satisfied with the lab (80% of TA preferred Y2 lab to Y1 lab; 73% were interested throughout the Y2 lab vs. 44% in Y1) and had more confidence in student's understanding of concepts (80% felt students understood respiration in Y2 vs. 72% in Y1; 80% felt students understood obstructive airway diseases in Y2 vs. 67% in Y1). Student quantitative assessment scores did not decrease during the study. Thus, in addition to receiving training on the lab itself, TA must be specifically instructed on the benefits of teaching a lab in an inquiry based fashion if they are to feel confident in their students' abilities during infusion of inquiry into a science classroom.