Structural Features Underlying the Multisite Phosphorylation of the A Domain of the NF-AT4 Transcription Factor by Protein Kinase CK1 †

ABSTRACT: The phosphorylation and dephosphorylation of the NF-AT family of transcription factors play a key role in the activation of T lymphocytes and in the control of the immune response. The mechanistic aspects of NF-AT4 phosphorylation by protein kinase CK1 have been studied in this work with the aid of a series of 27 peptides, reproducing with suitable modifications the regions of NF-AT4 that have been reported to be phosphorylated by this protein kinase. The largest parent peptide, representing the three regions A, Z, and L spanning amino acids 173-218, is readily phosphorylated by CK1 at seryl residues belonging to the A2 segment, none of which fulfill the canonical consensus sequence for CK1. An acidic cluster of amino acids in the linker region between domains A and Z is essential for high-efficiency phosphorylation of the A2 domain, as shown by the increase in K m caused by a deletion of the linker region or a substitution of the acidic residues with glycines. Individual substitutions with alanine of each of the five serines in the A2 domain (S-177, S-180, S-181, S-184, and S-186) reduce the phosphorylation rate, the most detrimental effect being caused by Ser177 substitution which results in a 10-fold drop in V max . On the contrary, the replacement of Ser177 with phosphoserine triggers a hierarchical effect with a dramatic improvement in phosphorylation efficiency, which no longer depends on the linker region for optimal efficiency. These data are consistent with a two-phase phosphorylation mechanism of NF-AT4 by CK1, initiated by the linker region which provides a functional docking site for CK1 and allows the unorthodox phosphorylation of Ser177; once achieved, this phosphoserine residue primes the phosphorylation of other downstream seryl residues, according to a hierarchical mechanism typically exploited by CK1. The large number of protein kinases in eukaryotes, with over 800 genes found in the human genome (1), raises multiple questions as to the function and specificity of these important enzymes. In recent years, several laboratories, including ours, have approached the study of the substrate specificity of protein kinases. These studies have concentrated on the analysis of the amino acid sequences surrounding the immediate vicinity of the sites that are phosphorylated in vivo and in vitro by specific kinases and on the preparation of synthetic peptides that contain these sequences and serve as substrates for these particular enzymes (2-5). These studies have been very useful in determining the consensus sequence recognized preferentially by the active center of these kinases and in predicting the domains of new proteins that are probably phosphorylated by these enzymes. In addition, this approach has allowed us to design several peptides that are highly specific for kinases and that can be employed in assaying for the activity of these kinases in crude extracts of cells and tissues (e.g., refs 5-7). The studies with short peptides, however, demonstrated that these model molecules are sometimes less efficient than the true physiological substrates. In addition, several sequences that contain the defined consensus for phosphorylation by these kinases are not phosphorylated in the native proteins. Conversely, atypical sites that are not acted upon in model peptides serve as good substrates within the context of whole proteins (5). These results clearly indicate that the phosphorylation of proteins by protein kinases involves recognition and interactions that go beyond the immediate vicinity of the acceptor serines or threonines in the substrates. The recent discovery that several protein kinases recognize "docking sites" which are distant from the phosphorylatable residues in their protein substrates constitutes an important step toward the understanding of some of the complexities that provide specificity in kinase-protein substrate interactions (8)

Anti-neoplastic drugs increase caveolin-1-dependent migration, invasion and metastasis of cancer cells

Indexación: Scopus.Expression of the scaffolding protein Caveolin-1 (CAV1) enhances migration and invasion of metastatic cancer cells. Yet, CAV1 also functions as a tumor suppressor in early stages of cancer, where expression is suppressed by epigenetic mechanisms. Thus, we sought to identify stimuli/mechanisms that revert epigenetic CAV1 silencing in cancer cells and evaluate how this affects their metastatic potential. We reasoned that restricted tissue availability of anti-neoplastic drugs during chemotherapy might expose cancer cells to sub-therapeutic concentrations, which activate signaling pathways and the expression of CAV1 to favor the acquisition of more aggressive traits. Here, we used in vitro [2D, invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question. Colon and breast cancer cells were identified where CAV1 levels were low due to epigenetic suppression and could be reverted by treatment with the methyltransferase inhibitor 5'-azacytidine. Exposure of these cells to anti-neoplastic drugs for short periods of time (24-48 h) increased CAV1 expression through ROS production and MEK/ERK activation. In colon cancer cells, increased CAV1 expression enhanced migration and invasion in vitro via pathways requiring Src-family kinases, as well as Rac-1 activity. Finally, elevated CAV1 expression in colon cancer cells following exposure in vitro to sub-cytotoxic drug concentrations increased their metastatic potential in vivo. Therefore exposure of cancer cells to anti-neoplastic drugs at non-lethal drug concentrations induces signaling events and changes in transcription that favor CAV1-dependent migration, invasion and metastasis. Importantly, this may occur in the absence of selection for drug-resistance.http://www.oncotarget.com/index.php?journal=oncotarget&page=article&op=view&path[]=22955&path[]=7243

Structural features underlying the multisite phosphorylation of the a domain of the NF-AT4 transcription factor by protein kinase CK1

The phosphorylation and dephosphorylation of the NF-AT family of transcription factors play a key role in the activation of T lymphocytes and in the control of the immune response. The mechanistic aspects of NF-AT4 phosphorylation by protein kinase CK1 have been studied in this work with the aid of a series of 27 peptides, reproducing with suitable modifications the regions of NF-AT4 that have been reported to be phosphorylated by this protein kinase. The largest parent peptide, representing the three regions A, Z, and L spanning amino acids 173-218, is readily phosphorylated by CK1 at seryl residues belonging to the A2 segment, none of which fulfill the canonical consensus sequence for CK1. An acidic cluster of amino acids in the linker region between domains A and Z is essential for high-efficiency phosphorylation of the A2 domain, as shown by the increase in Km caused by a deletion of the linker region or a substitution of the acidic residues with glycines. Individual substitution

Expression of a novel non-coding mitochondrial

RNA in human proliferating cell