Efectividad del ABP en asignaturas optativas de psicología

It is well documented that stimulation of cells by growth factors and cytokines results in activation of various protein tyrosine kinases that subsequently phosphorylates many protein substrates at tyrosine residue (Hunter and Cooper 1985). For instance, we previously demonstrated that PDGF or EGF stimulation led to tyrosine phosphorylation of p68 RNA helicase (Yang, Lin et al. 2005; Yang, Lin et al. 2006). Presently our experiments demonstrated that binding to tyrosine phosphor-protein converted PKM2 from tetramer to dimer therefore activated the protein kinase activity of the protein. We reason whether treatment of cells with growth factors would affect PKM2. Thus, SW480 cells were treated with EGF. The same chromatography procedure used for detecting the PKM2 tetramer vs dimer in crude cell extracts was employed here to analyze the tetramer and dimer ratio of PKM2 in cells under the stimulation of EGF. It was clear that the levels of dimer PKM2 in growth factor stimulated cells were significantly higher than that in corresponding unstimulated cells (Fig. 3.5A). Immunoblot analyses of PKM2 in nuclear and cytoplasmic extracts suggested that growth factor stimulation led to increase in nuclear PKM2 levels (Fig. 3.5B). Analyses of protein tyrosine phosphorylation and p68 tyrosine phosphorylation revealed that there was a substantial increase in protein tyrosine phosphorylation and tyrosine phosphorylation of p68 at tyrosine residue (Fig. 3.5C). These experiments suggested that indeed there is a clear correlation between the levels of protein tyrosine phosphorylations and dimeric PKM2 in cells, which is inducible by growth factors and cytokines.

During tumor progression, growth signals stimulate the conversion of glycolytically active PKM2 to an inactive form, consequently regulating the glycolysis pathway to channel the carbon source from glucose for biosynthesis (Deberardinis, 2008; Garber, 2006; Mazurek, 2003; Christofk, 2008). It is conceivable that tumor cells need to coordinate the metabolism alterations with expression of genes that are related to cell proliferation during tumor progression. Thus, at the same token, the growth signals will also activates protein kinase activity of PKM2, which subsequently target transcription factor and activate transcription of genes that closely associated with cell proliferation. PKM2 has long been recognized as a so-called ‘moonlight’ enzyme that plays a very important role in tumor progression (Sriram, 2005). Clearly, the protein kinase activity of PKM2 and subsequent activity in transcription regulation is an important ‘moonlight’ activity of the protein. The functions of PKM2 in regulating expression of genes fulfill the role of feedback signaling from metabolic alterations to gene regulation during tumor malignancy transformation (see model in Fig. 3.5D).

It is intriguing that a glycolytic enzyme can function as a protein kinase and translocate to the nucleus acting on gene transcription. Our studies suggested a molecular base for the activity conversion (Fig. 3.5D). Growth signal (growth factors or cytokines) stimulations lead to activation of protein tyrosine kinases, which phosphorylate a number of downstream targets. A number of tyrosine phosphor-proteins subsequently act on PKM2 by interacting with the protein at the FBP binding site. The tyrosine phosphor- protein and PKM2 interaction results in the conversion of PKM2 from tetramer to a dimer, which consequently lead to decrease in pyruvate kinase activity and increase in protein kinase activity. It was revealed from x-ray crystallography structural analyses of

the tetramer PKM2 that the ADP binding site is formed by a large hydrophobic hole, indicating a great flexibility for nucleotide binding (Muirhead, Clayden et al. 1986; Dombrauckas, Santarsiero et al. 2005). The large hydrophobic hole at the nucleotide binding site is almost completely buried in a tetramer structure, while it would be completely accessible with the dimeric form. Thus, it is conceivable that the nucleotide binding site may be able to accommodate a protein substrate when PKM2 is converted to a dimer. Alternatively, binding the tyrosine phosphor-protein to the FBP site would results in a significant conformation change that will allow PKM2 to accommodate a protein substrate binding. Stat3 is a transcription activator that is activated in response to inflammatory cytokines, such as IL-6 (Gao, 2007; Watson, 2008). Strikingly, activation of stat3 represents probably one of the most important molecular signatures involved in promoting cancer progression. It has been observed that activation of stat3 is detected in almost all cancer types (Frank 2007; Huang, Qiu et al. 2007; Kim, Chan et al. 2007; Groner, Lucks et al. 2008). However, it is generally believed that activations of stat3 in response to growth factor and cytokines are usually transient. A long standing question is “How do the malignant cancer cells maintain constitutive activation of stat3?” Currently, mutation(s) that lead to constitutive activation of stat3 have not been identified. Thus, activation of stat3 by PKM2 in malignant cancer cells potentially provides a very attractive explanation for this long-standing question. Whether stat3 is the only PKM2 substrate for its protein kinase activity is an open question. PKM2 interacts with several other proteins (Garcia-Gonzalo, Cruz et al. 2003; Lee, Kim et al. 2008; Spoden, Morandell et al. 2009). It was also reported that PKM2 purified from hepatoma tumour cells can phosphorylate histone H1 in vitro (Ignacak and Stachurska 2003). Thus, it will

be very interested to identify other PKM2 protein kinase substrates and uncover the putative cellular function of the corresponding protein phosphorylations.

3.5 Materials and Methods

3.5.1 Reagents, Cell lines, and Antibodies

The KLH conjugated peptide spans aa 399 – 412 of PKM2 (IYHLQLFEELRRLAPI) was synthesized by Global Peptide Services. PKM2 polycolonal antibody and anti-HA antibody was raised in the animal facility of Georgia State University. The peptides, D-PhoPepY593, PepY593, and PhosPepY567, were synthesized by AnaSpec. Antibodies against stat3, stat3 (pY705), HA-tag, GAPDH, Lamin A/C, and β-actin were purchased from Cell Signaling, Abnova, SantaCruz, Abcam, and AnaSpec respectively. Recombinant GST-stat3 was purchased from Abcam. Cell lines SW480, SW620, T98G were purchased from ATCC and cultured by following the vendor’s instructions.