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.

Oxygen deprivation or hypoxia characterizes a number of serious pathological conditions and elicits a number of adaptive changes that are mainly mediated at the transcriptional level by the family of hypoxia-inducible factors (HIFs). The HIF target gene repertoire includes genes responsible for the regulation of metabolism, oxygen delivery and cell survival. Although the involvement of HIFs in the regulation of carbohydrate metabolism and the switch to anaerobic glycolysis under hypoxia is well established, their role in the control of lipid anabolism and catabolism remains still relatively obscure. Recent evidence indicates that many aspects of lipid metabolism are modified during hypoxia or in tumor cells in a HIF-dependent manner, contributing significantly to the pathogenesis and/or progression of cancer and metabolic disorders. However, direct transcriptional regulation by HIFs has been only demonstrated in relatively few cases, leaving open the exact and isoform-specific mechanisms that underlie HIF-dependency. This review summarizes the evidence for both direct and indirect roles of HIFs in the regulation of genes involved in lipid metabolism as well as the involvement of HIFs in various diseases as demonstrated by studies with transgenic animal models.

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.

SummaryAdaptation to hypoxia involves hypoxia-inducible transcription factors (HIFs) and requires reprogramming of cellular metabolism that is essential during both physiological and pathological processes. In contrast to the established role of HIF-1 in glucose metabolism, the involvement of HIFs and the molecular mechanisms concerning the effects of hypoxia on lipid metabolism are poorly characterized. Here, we report that exposure of human cells to hypoxia causes accumulation of triglycerides and lipid droplets. This is accompanied by induction of lipin 1, a phosphatidate phosphatase isoform that catalyzes the penultimate step in triglyceride biosynthesis, whereas lipin 2 remains unaffected. Hypoxic upregulation of lipin 1 expression involves predominantly HIF-1, which binds to a single distal hypoxiaresponsive element in the lipin 1 gene promoter and causes its activation under low oxygen conditions. Accumulation of hypoxic triglycerides or lipid droplets can be blocked by siRNA-mediated silencing of lipin 1 expression or kaempferol-mediated inhibition of HIF-1. We conclude that direct control of lipin 1 transcription by HIF-1 is an important regulatory feature of lipid metabolism and its adaptation to hypoxia.

Hypoxia-inducible factor 1 (HIF-1) is the key transcriptional activator of hypoxia-inducible genes and an important anti-cancer target. Its regulated subunit, HIF-1␣, is controlled by oxygen levels and major signaling pathways. We reported previously that phosphorylation of Ser 641/643 by p42/44 MAPK is essential for HIF-1␣ nuclear accumulation and activity. We now show that a fragment of HIF-1␣ (amino acids 616 -658), termed MAPK target domain, contains a nuclear export signal (NES), which has atypical hydrophobic residue spacing. Localization, reporter gene, and co-immunoprecipitation assays demonstrate that the identified NES interacts with CRM1 in a phosphorylation-sensitive manner. Furthermore, disruption of the NES (I637A/L638A/I639A) restores nuclear localization and activity of nonphosphorylated HIF-1␣ and renders it largely resistant to inhibition of MAPK, an effect reproduced by a phosphomimetic mutation (S641E). As these data predict, overexpression of wildtype or mutant (S641A/S643A) MAPK target domain in HeLa cells modulates the activity and subcellular distribution of endogenous HIF-1␣. We suggest that control of HIF-1␣ nuclear transport represents an important MAPK-dependent regulatory mechanism.Hypoxia-inducible factor 1 (HIF-1) 2 is a transcriptional activator and the key mediator of cellular response to hypoxia. HIF-1 binds to regulatory DNA sequences called hypoxia-response elements and controls the expression of genes involved in cell metabolism, erythropoiesis, angiogenesis, invasion, and metastasis. It is therefore essential not only for cell survival under low oxygen but also for embryogenesis and tumor progression (1). HIF-1 is a heterodimer of HIF-1␣ and HIF-1␤ (or ARNT), both members of the basic helix-loop-helix Per-ARNT-Sim (PAS) family of transcription factors. Whereas ARNT is expressed constitutively, HIF-1␣ is highly regulated and a major target of anti-cancer therapy (2).Under normal oxygen concentration, HIF-1␣ protein levels are kept low by von Hippel-Lindau-mediated polyubiquitination and subsequent degradation. Interaction with von HippelLindau requires the hydroxylation of two proline residues in the oxygen-dependent degradation domain of HIF-1␣ (3). The proline hydroxylases that participate in this process depend on iron and require molecular oxygen and 2-oxoglutarate as substrates. When oxygen concentration is low, hydroxylation is impaired allowing HIF-1␣ to be stabilized, enter the nucleus, bind to ARNT and DNA, and induce the expression of target genes. This latter process is also regulated by oxygen tension as HIF-1␣ hydroxylation at Asn 803 by FIH-1 compromises its association with the transcriptional co-activator CBP/p300 (4). Reactive oxygen species produced under hypoxia as well as intermediate products of cell metabolism can also affect hydroxylation and, consequently, modulate HIF-1 activity (5-7).HIF-1␣ expression and transcriptional activity are additionally controlled, irrespective of oxygen levels, by major signaling pathways such as phosphatidylinositol 3-kin...

During mammalian spermiogenesis, histones are replaced by transition proteins, which are in turn replaced by protamines P1 and P2. P1 protamine contains a short arginine/serine-rich (RS) domain that is highly phosphorylated before being deposited into sperm chromatin and almost completely dephosphorylated during sperm maturation. We now demonstrate that, in elongating spermatids, this phosphorylation is required for the temporal association of P1 protamine with lamin B receptor (LBR), an inner nuclear membrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH 2 -terminal domain. Previous studies have shown that the cellular protein p32 also binds tightly to the unmodified RS domain of LBR. Extending those findings, we now present evidence that p32 prevents phosphorylation of LBR and furthermore that dissociation of this protein precedes P1 protamine association. Our data suggest that docking of protamine 1 to the nuclear envelope is an important intermediate step in spermiogenesis and reveal a novel role for SR protein kinases and p32.The development of spermatids into spermatozoa, termed spermiogenesis, is characterized by the replacement of histones by the highly basic, arginine-rich, protamines (1). As a result of this exchange, the nucleosomal-type chromatin is transformed into a smooth fiber and compacted in a volume of about 5% of that of a somatic cell nucleus (2, 3). Although the exchange of chromatin proteins during spermiogenesis has long been known, the molecular mechanisms and the signaling pathways governing the histone to protamine transition have remained obscure.The deposition of protamines on sperm chromatin and the subsequent chromatin condensation appear to be controlled by phosphorylation-dephosphorylation events. Protamines are highly phosphorylated, shortly after their synthesis and before binding to DNA, whereas they become largely dephosphorylated during sperm maturation (4 -8). Phosphorylation of P2 protamine has been shown to be essential, because deletion of the calmodulin-dependent protein kinase Camk4, which phosphorylates P2 protamine, impairs the replacement of transition protein-2 with P2 protamine, resulting in defective spermiogenesis and male sterility (9). On the other hand, all P1 protamines contain short arginine/serine-rich (RS) 1 domains that are efficiently phosphorylated by SRPK1 (SR protein kinase 1) (10), but the physiological significance of this modification is mostly unknown.In this respect, Biggiogera et al. (11) reported that protamines initially appear at the nuclear periphery, implying that the nuclear envelope might play a role in the replacement of transition proteins by protamines during spermiogenesis. Given that RS domains mediate protein-protein interactions (12), we sought to investigate the potential interaction of P1 protamine with the inner nuclear membrane protein lamin B receptor (LBR), which also possesses a repeat of RS dipeptides at its nucleoplasmic NH 2 -terminal domain. In the present study we demonstrate a direct a...

Hypoxia inducible factor-1 (HIF-1) is the main transcriptional activator of the cellular response to hypoxia and an important target of anticancer therapy. Phosphorylation by ERK1 and/or ERK2 (MAPK3 and MAPK1, respectively; hereafter ERK) stimulates the transcriptional activity of HIF-1α by inhibiting its CRM1 (XPO1)-dependent nuclear export. Here, we demonstrate that phosphorylation by ERK also regulates the association of HIF-1α with a so-far-unknown interaction partner identified as mortalin (also known as GRP75 and HSPA9), which mediates non-genomic involvement of HIF-1α in apoptosis. Mortalin binds specifically to HIF-1α that lacks modification by ERK, and the HIF-1α-mortalin complex is localized outside the nucleus. Under hypoxia, mortalin mediates targeting of unmodified HIF-1α to the outer mitochondrial membrane, as well as association with VDAC1 and hexokinase II, which promotes production of a C-terminally truncated active form of VDAC1, denoted VDAC1-ΔC, and protection from apoptosis when ERK is inactivated. Under normoxia, transcriptionally inactive forms of unmodified HIF-1α or its C-terminal domain alone are also targeted to mitochondria, stimulate production of VDAC1-ΔC and increase resistance to etoposide-or doxorubicin-induced apoptosis. These findings reveal an ERK-controlled, unconventional and anti-apoptotic function of HIF-1α that might serve as an early protective mechanism upon oxygen limitation and promote cancer cell resistance to chemotherapy.

Hypoxia-inducible factor 1 (HIF-1), a transcriptional activator that mediates cellular response to hypoxia and a promising target of anticancer therapy, is essential for adaptation to low oxygen conditions, embryogenesis and tumor progression. HIF-1 is a heterodimer of HIF-1α, expression of which is controlled by oxygen levels as well as by various oxygen-independent mechanisms, and HIF-1β (or ARNT), which is constitutively expressed. In this work, we investigate the phosphorylation of the N-terminal heterodimerization (PAS) domain of HIF-1α and identify Ser247 as a major site of in vitro modification by casein kinase 1δ (CK1δ). Mutation of this site to alanine, surprisingly, enhanced the transcriptional activity of HIF-1α, a result phenocopied by inhibition or small interfering RNA (siRNA)-mediated silencing of CK1δ under hypoxic conditions. Conversely, overexpression of CK1δ or phosphomimetic mutation of Ser247 to aspartate inhibited HIF-1α activity without affecting its stability or nuclear accumulation. Immunoprecipitation and in vitro binding experiments suggest that CK1-dependent phosphorylation of HIF-1α at Ser247 impairs its association with ARNT, a notion also supported by modeling the structure of the complex between HIF-1α and ARNT PAS-B domains. We suggest that modification of HIF-1α by CK1 represents a novel mechanism that controls the activity of HIF-1 during hypoxia by regulating the interaction between its two subunits.