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Hypoxia-inducible factor 1 (HIF-1) controls the expression of most genes induced by hypoxic conditions. Regulation of expression and activity of its inducible subunit, HIF-1␣, involves several post-translational modifications. To study HIF-1␣ phosphorylation, we have used human full-length recombinant HIF-1␣ as a substrate in kinase assays. We show that at least two different nuclear protein kinases, one of them identified as p42/p44 MAPK, can modify HIF-1␣. Analysis of in vitro phosphorylated HIF-1␣ by mass spectroscopy revealed residues Ser-641 and Ser-643 as possible MAPK phosphorylation sites. Site-directed mutagenesis of these residues reduced significantly the phosphorylation of HIF-1␣. When these mutant forms of HIF-1␣ were expressed in HeLa cells, they exhibited much lower transcriptional activity than the wild-type form. However, expression of the same mutants in yeast revealed that their capacity to stimulate transcription was not significantly compromised. Localization of the green fluorescent protein-tagged HIF-1␣ mutants in HeLa cells showed their exclusion from the nucleus in contrast to wild-type HIF-1␣. Treatment of the cells with leptomycin B, an inhibitor of the major exportin CRM1, reversed this exclusion and led to nuclear accumulation and partial recovery of the activity of the HIF-1␣ mutants. Moreover, inhibition of the MAPK pathway by PD98059 impaired the phosphorylation, nuclear accumulation, and activity of wild-type GFP-HIF-1␣. Overall, these data suggest that phosphorylation of Ser-641/643 by MAPK promotes the nuclear accumulation and transcriptional activity of HIF-1␣ by blocking its CRM1-dependent nuclear export.

History of the discovery of the serine-arginine protein kinase (SPRK) familyThe first serine-arginine (SR) protein kinase to be purified and characterized was named SRPK1, for SR-protein-specific kinase 1 [1,2]. It was isolated during a search for the activity that phosphorylates SR splicing factors (also named SR proteins) during mitosis. SRPK1 was shown to phosphorylate SR proteins in a cell-cycle regulated manner, to affect SR protein localization and to inhibit splicing when added in large quantities to a cell-free splicing assay [1,2]. The SRPK1 cDNA was cloned, revealing that the Schizosaccharomyces pombe SRPK1 orthologue, Dsk1, had already been cloned and partially characterized as a kinase with cell cycle-dependent phosphorylation and subcellular localization [3]. The SRPK1 and Dsk1 nucleotide sequencing identified a domain interrupting the kinase catalytic site into two structural entities, Serine-arginine protein kinases (SPRKs) constitute a relatively novel subfamily of serine-threonine kinases that specifically phosphorylate serine residues residing in serine-arginine ⁄ arginine-serine dipeptide motifs. Fifteen years of research subsequent to the purification and cloning of human SRPK1 as a SR splicing factor-phosphorylating protein have lead to the accumulation of information on the function and regulation of the different members of this family, as well as on the genomic organization of SRPK genes in several organisms. Originally considered to be devoted to constitutive and alternative mRNA splicing, SRPKs are now known to expand their influence to additional steps of mRNA maturation, as well as to other cellular activities, such as chromatin reorganization in somatic and sperm cells, cell cycle and p53 regulation, and metabolic signalling. Similarly, SRPKs were considered to be constitutively active kinases, although several modes of regulation of their function have been demonstrated, implying an elaborate cellular control of their activity. Finally, SRPK gene sequence information from bioinformatics data reveals that SRPK gene homologs exist either in single or multiple copies in every single eukaryotic organism tested, emphasizing the importance of SRPK protein function for cellular life.Abbreviations CDK, cyclin dependent kinase; Clk, CDK-like kinase; CK2, casein kinase 2; FOXO1, forkhead box protein O1; HBV, hepatitis B virus; HP1, heterochromatin protein 1; Hsp, heat shock protein; LBR, lamin B receptor; NRF-1, nuclear respiratory factor-1; PGC-1, peroxisome proliferator activated receptor c coactivator-1; RS, arginine-serine; SAFB, scaffold attachment factor B; SR, serine-arginine; SRPK, serine-arginine protein kinase.

Iron uptake in Saccharomyces cerevisiae involves at least two steps: reduction of ferric to ferrous ions extracellularly and transport of the reduced ions through the Iron is an indispensable element for living organisms. Oxygen storage and transport in plants and animals (leghemoglobins, hemoglobin, myoglobin, and hemerythrin), respiration, photosynthesis and electron transport (cytochromes), and nucleic acid metabolism (ribonucleotide reductase) are some examples of biological functions carried out principally by iron-containing proteins.Two features render iron an arduous metal to be handled by living organisms. The first is its availability; although it is the second most abundant metal in the earth's crust, iron is found primarily in the ferric (Fe3") state, forming hydroxides or salts of very low solubility and thus biologically inaccessible by simple mechanisms. The second is its toxicity; iron, in conjunction with oxygen, is a generator of hydroxyl radicals, which have a variety of toxic effects on cells.Living organisms as diverse as prokaryotes and mammals have developed a variety of mechanisms to overcome the problem of iron bioavailability as well as to regulate the iron concentration in biological fluids and cell compartments, in order to ensure its proper function as opposed to its toxic effects (reviewed in references 7 and 16). Two principal mechanisms have been described for iron uptake. One involves the use of iron chelators, either small molecules such as the siderophores secreted by bacteria, fungi, and plants or polypeptides such as transferrin and lactoferrin which are found in biological fluids of higher eukaryotes. All of these molecules keep ferric ions in a soluble form and deliver them to the cells mainly by receptor-mediated endocytosis. The second mechanism for iron uptake involves an initial reduction step in the external vicinity of the plasma membrane followed by internalization of the ferrous ions by means of ion transporters.Membrane-associated ferric reductase activity has been de-* Corresponding author. Mailing address: