Steroid hormones may regulate autophosphorylation of adenosine-3',5'-monophosphate-dependent protein kinase in target tissues

A protein in rat liver cytosol whose phosphorylation was regulated by hydrocortisone administration in vivo was tentatively identified as the regulatory subunit of a cAMP-dependent protein kinase. Evidence that this protein, whose phosphorylation was regulated by steroid and cyclic AMP, is the regulatory subunit of type-II cAMP-dependent protein kinase included: (a) co-purification of the steroid/cAMP-regulated protein and the regulatory subunit during DEAE-cellulose, Sepharose 4B, and hydroxylapatite column chromatography, (b) co-migration of the two proteins on dodecyl sulfate/polyacrylamide slab gels during the various steps of purification, (c) specific adsorption of the two proteins onto 8(6-aminohexylamino)-cAMP--Sepharose 4B, and (d) a similar pattern of distribution of the two proteins in various subcellular fractions prepared from rat liver homogenate. By each of these criteria, it was found that the steroid/cAMP-regulated protein present in rat liver cytosol behaved identically with the regulatory subunit of type-II cAMP-dependent protein kinase in that tissue. Results qualitatively similar to those obtained in the study of the effect of hydrocortisone on rat liver were also obtained in studies of the effects of other steroid hormones on other target tissues in the rat, including uterus (17 beta-estradiol), ventral prostate and seminal vesicle (testosterone), and epididymal fat pad (hydrocortisone). The tentative identification of the steroid/cAMP-regulated protein as the regulatory subunit of the type-II cAMP-dependent protein kinase in the cytosol of several tissues indicates that autophosphorylation of the regulatory subunit of type-II protein kinase may be regulated by the steroid hormones. The fact that three different classes of steroid hormones appear to affect the phosphorylation of the regulatory subunit of type-II cAMP-dependent protein kinase in their target tissues raises the possibility that this common biochemical action may play an important role in the mechanism of steroid hormone action. It is also possible that this effect of the steroid hormones may provide a molecular basis for some of the known physiological interactions of the steroid hormones with those hormones that act through using cAMP as a second messenger.     

Interaction of cCMP with the cGK,cAK and MAPK Kinases in Murine Tissues

cAMP and cGMP are well established second messengers that are essential for numerous (patho)physiological processes. These purine cyclic nucleotides activate cAK and cGK, respectively. Recently, the existence of cCMP was described, and a possible function for this cyclic nucleotide was investigated. It was postulated that cCMP plays a role as a second messenger. However, the functions regulated by cCMP are mostly unknown. To elucidate probable functions, cCMP-binding and -activated proteins were identified using different methods. We investigated the effect of cCMP on purified cyclic nucleotide-dependent protein kinases and lung and jejunum tissues of wild type (WT), cGKI-knockout (cGKI KO) and cGKII-knockout (cGKII KO) mice. The catalytic activity of protein kinases was measured by a (γ-³²P) ATP kinase assay. Cyclic nucleotide-dependent protein kinases (cAK, cGKI and cGKII) in WT tissue lysates were stimulated by cCMP. In contrast, there was no stimulation of phosphorylation in KO tissue lysates. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. These complexes were abolished or reduced in jejunum tissues from cGKI KO or cGKII KO mice. In contrast, these complexes were observed in the lung tissues from WT, cGKI KO and cGKII KO mice. Moreover, cCMP was also able to stimulate the phosphorylation of MAPK. These results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Based on these results, we propose that additional cCMP-dependent protein kinases that are capable of modulating MAPK signaling could exist. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum and lung.     

Quantitative proteomic comparison of rat mitochondria from muscle, heart, and liver

Mitochondria, through oxidative phosphorylation, are the primary source of energy production in all tissues under aerobic conditions. Although critical to life, energy production is not the only function of mitochondria, and the composition of this organelle is tailored to meet the specific needs of each cell type. As an organelle, the mitochondrion has been a popular subject for proteomic analysis, but quantitative proteomic methods have yet to be applied to tease apart subtle differences among mitochondria from different tissues or muscle types. Here we used mass spectrometry-based proteomics to analyze mitochondrial proteins extracted from rat skeletal muscle, heart, and liver tissues. Based on 689 proteins identified with high confidence, mitochondria from the different tissues are qualitatively quite similar. However, striking differences emerged from the quantitative comparison of protein abundance between the tissues. Furthermore we applied similar methods to analyze mitochondrial matrix and intermembrane space proteins extracted from the same mitochondrial source, providing evidence for the submitochondrial localization of a number of proteins in skeletal muscle and liver. Several proteins not previously thought to reside in mitochondria were identified, and their presence in this organelle was confirmed by protein correlation profiling. Hierarchical clustering of microarray expression data provided further evidence that some of the novel mitochondrial candidates identified in the proteomic survey might be associated with mitochondria. These data reveal several important distinctions between mitochondrial and submitochondrial proteomes from skeletal muscle, heart, and liver tissue sources. Indeed approximately one-third of the proteins identified in the soluble fractions are associated predominantly to one of the three tissues, indicating a tissue-dependent regulation of mitochondrial proteins. Furthermore a small percentage of the mitochondrial proteome is unique to each tissue.     

Phosphorylation of IRS proteins, insulin action, and insulin resistance

Insulin signaling at target tissues is essential for growth and development and for normal homeostasis of glucose, fat, and protein metabolism. Control over this process is therefore tightly regulated. It can be achieved by a negative feedback control mechanism whereby downstream components inhibit upstream elements along the insulin-signaling pathway (autoregulation) or by signals from apparently unrelated pathways that inhibit insulin signaling thus leading to insulin resistance. Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues has emerged as a key step in these control processes under both physiological and pathological conditions. The list of IRS kinases implicated in the development of insulin resistance is growing rapidly, concomitant with the list of potential Ser/Thr phosphorylation sites in IRS proteins. Here, we review a range of conditions that activate IRS kinases to phosphorylate IRS proteins on "hot spot" domains. The flexibility vs. specificity features of this reaction is discussed and its characteristic as an "array" phosphorylation is suggested. Finally, its implications on insulin signaling, insulin resistance and type 2 diabetes, an emerging epidemic of the 21st century are outlined.     

AFAP120 regulates actin organization during neuronal differentiation

During development, dynamic changes in the actin cytoskeleton determine both cell motility and morphological differentiation. In most mature tissues, cells are generally minimally motile and have morphologies specialized to their functions. In metastatic cancer, cells generally lose their specialized morphology and become motile. Therefore, proteins that regulate the transition between the motile and morphologically differentiated states can play important roles in determining cancer outcomes. AFAP120 is a neuronal-specific protein that binds Src kinase and protein kinase C (PKC) and cross-links actin filaments. Here we report that expression and tyrosine phosphorylation of AFAP120 are developmentally regulated in the cerebellum. In cerebellar cultures, PKC activation induces Src kinase-dependent phosphorylation of AFAP120, indicating that AFAP120 may be a downstream effector of Src. In neuroblastoma cells induced to differentiate by treatment with a PKC activator, tyrosine phosphorylation of AFAP120 appears to regulate the formation of the lamellar actin structures and subsequent neurite initiation. Together, these results indicate that AFAP120 plays a role in organizing dynamic actin structures during neuronal differentiation and suggest that AFAP120 may help regulate the transition from motile precursor to morphologically differentiated neurons.     

ISG15, not just another ubiquitin-like protein

ISG15 is a ubiquitin-like protein containing two ubiquitin homology domains and becomes conjugated to a variety of proteins when cells are treated with type I interferon or lipopolysaccharide. Although ISG15 shares several common properties with those of other ubiquitin-like molecules, it is a unique member, whose expression and conjugation to target proteins are tightly regulated by specific signaling pathways, indicating it may be associated with specialized functions in innate immune system. Loss of UBP43 (USP18), a protease that specifically removes ISG15 from ISG15-modified proteins, in mice leads to decreased life span, brain cell injury, and hypersensitivity to interferon stimulation. In UBP43 deficient cells, interferon induces a prolonged Stat1 tyrosine phosphorylation and DNA binding, which result in a prolonged and enhanced activation of interferon-stimulated genes.     

A global view of protein expression in human cells,tissues, and organs

Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high‐resolution, immunohistochemistry‐based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced.     

The PP1 phosphatase flapwing regulates the activity of Merlin and Moesin in Drosophila

The signalling activities of Merlin and Moesin, two closely related members of the protein 4.1 Ezrin/Radixin/Moesin family, are regulated by conformational changes. These changes are regulated in turn by phosphorylation. The same sterile 20 kinase-Slik co-regulates Merlin or Moesin activity whereby phosphorylation inactivates Merlin, but activates Moesin. Thus, the corresponding coordinate activation of Merlin and inactivation of Moesin would require coordinated phosphatase activity. We find that Drosophila melanogaster protein phosphatase type 1 β (flapwing) fulfils this role, co-regulating dephosphorylation and altered activity of both Merlin and Moesin. Merlin or Moesin are detected in a complex with Flapwing both in-vitro and in-vivo. Directed changes in flapwing expression result in altered phosphorylation of both Merlin and Moesin. These changes in the levels of Merlin and Moesin phosphorylation following reduction of flapwing expression are associated with concomitant defects in epithelial integrity and increase in apoptosis in developing tissues such as wing imaginal discs. Functionally, the defects can be partially recapitulated by over expression of proteins that mimic constitutively phosphorylated or unphosphorylated Merlin or Moesin. Our results suggest that changes in the phosphorylation levels of Merlin and Moesin lead to changes in epithelial organization.     

An integrative proteome analysis of different seedling organs in tolerant and sensitive wheat cultivars under drought stress and recovery

Roots, leaves, and intermediate sections between roots and leaves (ISRL) of wheat seedlings show different physiological functions at the protein level. We performed the first integrative proteomic analysis of different tissues of the drought‐tolerant wheat cultivar Hanxuan 10 (HX‐10) and drought‐sensitive cultivar Chinese Spring (CS) during a simulated drought and recovery. Differentially expressed proteins (DEPs) in the roots (122), ISRLs (146), and leaves (163) showed significant changes in expression in response to drought stress and recovery. Numerous DEPs associated with cell defense and detoxifications were significantly regulated in roots and ISRLs, while in leaves, DEPs related to photosynthesis showed significant changes in expression. A significantly larger number of DEPs related to stress defense were upregulated in HX‐10 than in CS. Expression of six HSPs potentially related to drought tolerance was significantly upregulated under drought conditions, and these proteins were involved in a complex protein–protein interaction network. Further phosphorylation analysis showed that the phosphorylation levels of HSP60, HSP90, and HOP were upregulated in HX‐10 under drought stress. We present an overview of metabolic pathways in wheat seedlings based on abscisic acid signaling and important protein expression patterns.     

Genomic DNA microarray analysis: identification of new genes regulated by light color in the cyanobacterium Fremyella diplosiphon

Many cyanobacteria use complementary chromatic adaptation to efficiently utilize energy from both green and red regions of the light spectrum during photosynthesis. Although previous studies have shown that acclimation to changing light wavelengths involves many physiological responses, research to date has focused primarily on the expression and regulation of genes that encode proteins of the major photosynthetic light-harvesting antennae, the phycobilisomes. We have used two-dimensional gel electrophoresis and genomic DNA microarrays to expand our understanding of the physiology of acclimation to light color in the cyanobacterium Fremyella diplosiphon. We found that the levels of nearly 80 proteins are altered in cells growing in green versus red light and have cloned and positively identified 17 genes not previously known to be regulated by light color in any species. Among these are homologs of genes present in many bacteria that encode well-studied proteins lacking clearly defined functions, such as tspO, which encodes a tryptophan-rich sensory protein, and homologs of genes encoding proteins of clearly defined function in many species, such as nblA and chlL, encoding phycobilisome degradation and chlorophyll biosynthesis proteins, respectively. Our results suggest novel roles for several of these gene products and highly specialized, unique uses for others.     

Method development of efficient protein extraction in bone tissue for proteome analysis

Exploring bone proteome is an important and challenging task for understanding the mechanisms of physiological/pathological process of bone tissue. However, classical methods of protein extraction for soft tissues and cells are not applicable for bone tissue. Therefore, method development of efficient protein extraction is critical for bone proteome analysis. We found in this study that the protein extraction efficiency was improved significantly when bone tissue was demineralized by hydrochloric acid (HCl). A sequential protein extraction method was developed for large-scale proteome analysis of bone tissue. The bone tissue was first demineralized by HCl solution and then extracted using three different lysis buffers. As large amounts of acid soluble proteins also presented in the HCl solution, besides collection of proteins in the extracted lysis buffers, the proteins in the demineralized HCl solution were also collected for proteome analysis. Automated 2D-LC-MS/MS analysis of the collected protein fractions resulted in the identification of 6202 unique peptides which matched 2479 unique proteins. The identified proteins revealed a broad diversity in the protein identity and function. More than 40 bone-specific proteins and 15 potential protein biomarkers previously reported were observed in this study. It was demonstrated that the developed extraction method of proteins in bone tissue, which was also the first large-scale proteomic study of bone, was very efficient for comprehensive analysis of bone proteome and might be helpful for clarifying the mechanisms of bone diseases.     

Expression of phosphophoryn is sufficient for the induction of matrix mineralization by mammalian cells

Mineralized tissues such as dentin and bone assemble extracellular matrices uniquely rich in a variety of acidic phosphoproteins. Although these proteins are presumed to play a role in the process of biomineralization, key questions regarding the nature of their contributions remain unanswered. First, it is not known whether highly phosphorylated proteins alone can induce matrix mineralization, or whether this activity requires the involvement of other bone/dentin non-collagenous proteins. Second, it remains to be established whether the protein kinases that phosphorylate these acidic proteins are unique to cells responsible for producing mineralized tissues. To begin to address these questions, we consider the case of phosphophoryn (PP), due to its high content of phosphate, high affinity for Ca(2+), and its potential role in hydroxyapatite nucleation. We have created a model system of biomineralization in a cellular environment by expressing PP in NIH3T3 fibroblasts (which do not produce a mineralized matrix); as a positive control, PP was expressed in MC3T3-E1 osteoblastic cells, which normally mineralize their matrices. We show that expression of PP in NIH3T3 cells is sufficient for the induction of matrix mineralization. In addition, assessment of the phosphorylation status of PP in these cells reveals that the transfected NIH3T3 cells are able to phosphorylate PP. We suggest that the phosphorylation of PP is essential for mineral formation. The principle goal of this study is to enrich the current knowledge of mineralized tissue phosphorylation events by analyzing them in the context of a complete cellular environment.     

CDC2-related kinase PITALRE phosphorylates pRb exclusively on serine and is widely expressed in human tissues

Mammalian cell cycle progression is regulated by sequential activation and inactivation of cyclin-dependent kinases (cdks). Recently, several new members of the cdk family were cloned, and some of these were shown to complex with different cyclins and to be active at discrete stages of the cell cycle. PITALRE, a new member of this family, was cloned by our laboratory and was shown to be able to phosphorylate pRb protein in vitro. In the current work, we found that PITALRE kinase activity phosphorylated pRb at sites similar to those phosphorylated by the CDC2 kinase, which itself is known to mimic, in vitro, the in vivo phosphorylation of pRb. Phosphorylation of pRb by the PITALRE-associated kinase activity was on Ser residues exclusively. Moreover, we investigated the expression pattern of PITALRE in normal human tissues, using immunohistochemical techniques so as to gain additional data on the characteristics of this new cdk family member. The protein was widely expressed, although a different tissue distribution and/or level of expression was found in various organs. Some specialized tissues such as blood, lymphoid tissue, ovarian cells, and the endocrine portion of the pancreas showed a high expression level of PITALRE. The specific expression pattern found suggests that PITALRE may be involved in specialized functions in certain cell types. J. Cell. Physiol. 172:265–273, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of post-translational modifications of muskelin by protein kinase C

Muskelin is a member of the kelch-repeat superfamily of proteins, identified as an intracellular protein involved in cell spreading responses to thombospondin-1. Muskelin is expressed by many adult tissues and has an evolutionarily conserved, multidomain architecture consisting of an amino-terminal discoidin-like domain, a central alpha-helical region and six kelch-repeats that are predicted to form a beta-propeller structure. We previous demonstrated that muskelin molecules undergo head-to-tail association, however the physiological, post-translational regulation of muskelin is not well understood. Here, we have examined the expression of muskelin during mouse embryonic development and report widespread expression that includes muscle tissues, multiple epithelia and the brain. In cultured skeletal myoblasts and vascular smooth muscle cells, muskelin exists as a complex set of isoelectric variants. Five potential sites for phosphorylation by protein kinase C (PKC), are conserved between vertebrate and Drosophila muskelins, therefore we examined the hypothesis that muskelin is regulated post-translationally by PKC activity. We demonstrate that PKC activation or inhibition regulates the profile of endogenous muskelin isoelectric variants and that muskelin is a substrate for PKCalphain vitro. Wild-type GFP-muskelin and a panel of alanine point mutations were used to test the sensitivity of self-association to PKC activation. Mutation of two of the sites, S324 and T515, partially inhibited the ability of muskelin to self-associate in cells and inhibited responsiveness to activated PKC. Interestingly, both sites are predicted to lie in surface-exposed loops on the same side of the beta-propeller, implicating a common binding interface.     

The effects of connexin phosphorylation on gap junctional communication

Gap junctions are specialized membrane domains composed of collections of channels that directly connect neighboring cells providing for the cell-to-cell diffusion of small molecules, including ions, amino acids, nucleotides, and second messengers. Vertebrate gap junctions are composed of proteins encoded by the "connexin" gene family. In most cases examined, connexins are modified post-translationally by phosphorylation. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the connexin "lifecycle", such as the trafficking, assembly/disassembly, degradation, as well as, the gating of gap junction channels. Since connexin43 (Cx43) is widely expressed in tissues and cell lines, we understand the most about how it is regulated, and thus, connexin43 phosphorylation is a major focus of this review. Recent reports utilizing new methodologies combined with the latest genome information have shown that activation of several kinases including protein kinase A, protein kinase C, p34(cdc2)/cyclin B kinase, casein kinase 1, mitogen-activated protein (MAP) kinase and pp60(src) kinase can lead to phosphorylation at 12 of the 21 serine and two of the six tyrosine residues in the C-terminal region of connexin43. In several cases, use of site-directed mutants of these sites have shown that these specific phosphorylation events can be linked to changes in gap junctional communication.     

Eukaryotic translation initiation factor 4E (eIF4E) expression in the brain tissue is induced by infusion of nerve growth factor into the mouse cisterna magnum: an in vivo study

In many cell types translation can be regulated by an expression of the translation initiation factor. Eukaryotic translation initiation factor eIF4E, which binds to the 5′ cap structure of mRNA, plays an important role in translation regulation and it has been suggested that it is implicated in increased protein synthesis promoted by growth factors. In this study the effects of nerve growth factor (NGF) infusion into the cerebrospinal fluid (CSF) on eIF4E expression and phosphorylation in mouse brain tissue have been investigated. We investigated NGF as it is one of the most important growth factors and it is an important factor in cerebral cortical development, stimulating neuronal precursor proliferation. eIF4E level is also increased in response to infusion of NGF into the CSF. The present study shows that eIF4E is phosphorylated in the brain tissues treated with NGF. It is concluded that NGF regulates protein synthesis in the nervous tissue by enhancing expression and phosphorylation of eIF4E.