Protein phosphatase 4 is a positive regulator of hematopoietic progenitor kinase 1

Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic specific mammalian Ste20-like protein kinase and has been implicated in many cellular signaling pathways including T cell receptor (TCR) signaling. However, little is known about the in vivo regulation of HPK1. We present evidence that HPK1 is positively regulated by protein phosphatase 4 (PP4; also called PPX and PPP4), a serine/threonine phosphatase. We found that PP4 interacted with HPK1 and that the proline-rich region of HPK1 was necessary and sufficient for this interaction. We also found that PP4 had phosphatase activity toward HPK1 in vivo and that co-transfection of PP4 with HPK1 resulted in specific kinase activation of HPK1. Moreover, we found that the PP4-induced HPK1 kinase activation was accompanied by an increase in protein expression of HPK1. Pulse-chase analysis showed that PP4 increased the half-life of HPK1. Further studies showed that HPK1 was subject to regulation by ubiquitination and ubiquitin-targeted degradation and that PP4 inhibited HPK1 ubiquitination. In addition, we found that TCR stimulation enhanced the PP4-HPK1 interaction and that wild-type PP4 enhanced, whereas a phosphatase-dead PP4 mutant inhibited, TCR-induced activation of HPK1 in Jurkat T cells. Combined with the observation that PP4 enhanced HPK1-induced JNK activation, our studies identify PP4 as a positive regulator for HPK1 and the HPK1-JNK signaling pathway.     

Protein phosphatase 2A (PP2A)-specific ubiquitin ligase MID1 is a sequence-dependent regulator of translation efficiency controlling 3-phosphoinositide-dependent protein kinase-1 (PDPK-1)

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells.     

Usage of tautomycetin,a novel inhibitor of protein phosphatase 1 (PP1), reveals that PP1 is a positive regulator of Raf-1 in vivo

Protein phosphatase type 1 (PP1), together with protein phosphatase 2A (PP2A), is a major eukaryotic serine/threonine protein phosphatase involved in regulation of numerous cell functions. Although the roles of PP2A have been studied extensively using okadaic acid, a well known inhibitor of PP2A, biological analysis of PP1 has remained restricted because of lack of a specific inhibitor. Recently we reported that tautomycetin (TC) is a highly specific inhibitor of PP1. To elucidate the biological effects of TC, we demonstrated in preliminary experiments that treatment of COS-7 cells with 5 microm TC for 5 h inhibits endogenous PP1 by more than 90% without affecting PP2A activity. Therefore, using TC as a specific PP1 inhibitor, the biological effect of PP1 on MAPK signaling was examined. First, we found that inhibition of PP1 in COS-7 cells by TC specifically suppresses activation of ERK, among three MAPK kinases (ERK, JNK, and p38). TC-mediated inhibition of PP1 also suppressed activation of Raf-1, resulting in the inactivation of the MEK-ERK pathway. To examine the role of PP1 in regulation of Raf-1, we overexpressed the PP1 catalytic subunit (PP1C) in COS-7 cells and found that PP1C enhanced activation of Raf-1 activity, whereas phosphatase-dead PP1C blocked Raf-1 activation. Furthermore, a physical interaction between PP1C and Raf-1 was also observed. These data strongly suggest that PP1 positively regulates Raf-1 in vivo.     

Multiscale complexity in the mammalian circadian clock

The field of systems biology studies how the interactions among individual components (e.g. genes and proteins) yield interesting and complex behavior. The circadian (daily) timekeeping system in mammals is an ideal system to study complexity because of its many biological scales (from genes to animal behavior). A wealth of data at each of these scales has recently been discovered. Within each scale, modeling can advance our understanding of challenging problems that arise in studying mammalian timekeeping. However, future work must focus on bridging the multiple spatial and temporal scales in the modeling of SCN network. Here we review recent advances, and then delve into a few areas that are promising research directions. We also discuss the flavor of modeling needed (simple or detailed) as well as new techniques that are needed to meet the challenges in modeling data across scales.     

Protein phosphatase with EF-hand domains 2 (PPEF2) is a potent negative regulator of apoptosis signal regulating kinase-1 (ASK1)

The function of protein phosphatases with EF-hand domains (PPEF) in mammals is not known. Large-scale expression profiling experiments suggest that PPEF expression may correlate with stress protective responses, cell survival, growth, proliferation, or neoplastic transformation. Apoptosis signal regulating kinase-1 (ASK1) is a MAP kinase kinase kinase implicated in cancer, cardiovascular and neurodegenerative diseases. ASK1 is activated by oxidative stress and induces pro-apoptotic or inflammatory signalling, largely via sustained activation of MAP kinases p38 and/or JNK. We identify human PPEF2 as a novel interacting partner and a negative regulator of ASK1. In COS-7 or HEK 293A cells treated with H₂O₂, expression of PPEF2 abrogated sustained activation of p38 and one of the JNK p46 isoforms, and prevented ASK1-dependent caspase-3 cleavage and activation. PPEF2 efficiently suppressed H₂O₂-induced activation of ASK1. Overexpessed as well as endogenous ASK1 co-immunoprecipitated with PPEF2. PPEF2 was considerably more potent both as a suppressor of ASK1 activation and as its interacting partner as compared to protein phosphatase 5 (PP5), a well-known negative regulator of ASK1. PPEF2 was found to form complexes with endogenous Hsp70 and to a lesser extent Hsp90, which are also known interacting partners of PP5. These data identify, for the first time, a possible downstream signalling partner of a mammalian PPEF phosphatase, and suggest that, despite structural divergence, PPEF and PP5 phosphatases may share common interacting partners and functions.     

Kinases and phosphatases in the mammalian circadian clock

Posttranslational modifications of circadian oscillator components are crucial for the generation of circadian rhythms. Among those phosphorylation plays key roles ranging from regulating degradation, complex formation, subcellular localization and activity. Although most of the known clock proteins are phosphoproteins in vivo, a comprehensive view about the regulation of clock protein phosphorylation is still missing. Here, we review our current knowledge about the role of clock protein phosphorylation and its regulation by kinases and phosphatases in eukaryotes with a major focus on the mammalian circadian clock.     

The circadian clock system in the mammalian retina

Daily rhythms are a ubiquitous feature of living systems. Generally, these rhythms are not just passive consequences of cyclic fluctuations in the environment, but instead originate within the organism. In mammals, including humans, the master pacemaker controlling 24-hour rhythms is localized in the suprachiasmatic nuclei of the hypothalamus. This circadian clock is responsible for the temporal organization of a wide variety of functions, ranging from sleep and food intake, to physiological measures such as body temperature, heart rate and hormone release. The retinal circadian clock was the first extra-SCN circadian oscillator to be discovered in mammals and several studies have now demonstrated that many of the physiological, cellular and molecular rhythms that are present within the retina are under the control of a retinal circadian clock, or more likely a network of hierarchically organized circadian clocks that are present within this tissue. BioEssays 30:624-633, 2008. (c) 2008 Wiley Periodicals, Inc.