Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3 beta phosphorylation

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating beta-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization [1, 2]. The microtubule binding domain is deleted in the transforming mutations of APC [3, 4]; however, the direct effect of APC protein on microtubules has never been examined. Here we show that binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3 beta. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation.     

Regulation of the autophagy protein LC3 by phosphorylation

Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP⁺) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease–associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl–cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity.     

Regulation of PTEN phosphorylation and stability by a tumor suppressor candidate protein

The tumor suppressor PTEN plays an essential role in regulating signaling pathways involved in cell growth and apoptosis and is inactivated in a wide variety of tumors. In this study, we have identified a protein, referred to as PICT-1 (protein interacting with carboxyl terminus 1), that binds to the C terminus of PTEN and regulates its phosphorylation and turnover. Down-regulation of PICT-1 in MCF7 cells by RNA interference enhances the degradation of PTEN with a concomitant decrease in its phosphorylation. PTEN C-terminal tumor-associated mutants, which are highly susceptible to protein degradation, have lost the ability to bind to PICT-1 along with their reduced phosphorylation, suggesting that their rapid turnover results from impaired binding to PICT-1. Our results identify PICT-1 as a PTEN-interacting protein that promotes the phosphorylation and stability of PTEN. These findings suggest a novel molecular mechanism underlying the turnover of PTEN, which also provides an explanation for the loss of PTEN function due to C-terminal mutations.     

Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3

NDR protein kinases are involved in the regulation of cell cycle progression and morphology. NDR1/NDR2 protein kinase is activated by phosphorylation on the activation loop phosphorylation site Ser281/Ser282 and the hydrophobic motif phosphorylation site Thr444/Thr442. Autophosphorylation of NDR is responsible for phosphorylation on Ser281/Ser282, whereas Thr444/Thr442 is targeted by an upstream kinase. Here we show that MST3, a mammalian Ste20-like protein kinase, is able to phosphorylate NDR protein kinase at Thr444/Thr442. In vitro, MST3 selectively phosphorylated Thr442 of NDR2, resulting in a 10-fold stimulation of NDR activity. MOB1A (Mps one binder 1A) protein further increased the activity, leading to a fully active kinase. In vivo, Thr442 phosphorylation after okadaic acid stimulation was potently inhibited by MST3KR, a kinase-dead mutant of MST3. Knockdown of MST3 using short hairpin constructs abolished Thr442 hydrophobic motif phosphorylation of NDR in HEK293F cells. We conclude that activation of NDR is a multistep process involving phosphorylation of the hydrophobic motif site Thr444/2 by MST3, autophosphorylation of Ser281/2, and binding of MOB1A.     

Regulation of actin function by protein kinase A‐mediated phosphorylation of Limk1

Proper regulation of the cAMP-dependent protein kinase (protein kinase A, PKA) is necessary for cellular homeostasis, and dysregulation of this kinase is crucial in human disease. Mouse embryonic fibroblasts (MEFs) lacking the PKA regulatory subunit Prkar1a show altered cell morphology and enhanced migration. At the molecular level, these cells showed increased phosphorylation of cofilin, a crucial modulator of actin dynamics, and these changes could be mimicked by stimulating the activity of PKA. Previous studies of cofilin have shown that it is phosphorylated primarily by the LIM domain kinases Limk1 and Limk2, which are under the control of the Rho GTPases and their downstream effectors. In Prkar1a^−/− MEFs, neither Rho nor Rac was activated; rather, we showed that PKA could directly phosphorylate Limk1 and thus enhance the phosphorylation of cofilin. These data indicate that PKA is crucial in cell morphology and migration through its ability to modulate directly the activity of LIM kinase.     

Regulation of sprouty stability by Mnk1-dependent phosphorylation

Sprouty (Spry) proteins are negative feedback modulators of receptor tyrosine kinase pathways in Drosophila melanogaster and mammals. Mammalian Spry proteins have been shown to undergo tyrosine and serine phosphorylation in response to growth factor stimulation. While several studies have addressed the function of tyrosine phosphorylation of Spry, little is known about the significance of Spry serine phosphorylation. Here we identify mitogen-activated protein kinase-interacting kinase 1 (Mnk1) as the kinase that phosphorylates human Spry2 (hSpry2) on serines 112 and 121. Mutation of these serine residues to alanine or inhibition of Mnk1 activity increases the rate of ligand-induced degradation of hSpry2. Conversely, enhancement of serine phosphorylation achieved through either the inhibition of cellular phosphatases or the expression of active Mnk1 results in the stabilization of hSpry2. Previous studies have shown that growth factor stimulation induces the proteolytic degradation of hSpry2 by stimulating tyrosine phosphorylation on hSpry2, which in turn promotes c-Cbl binding and polyubiquitination. A mutant of hSpry2 that is deficient in serine phosphorylation displays enhanced tyrosine phosphorylation and c-Cbl binding, indicating that serine phosphorylation stabilizes hSpry2 by exerting an antagonistic effect on tyrosine phosphorylation. Moreover, loss of serine phosphorylation and the resulting enhanced degradation of hSpry2 impair its capacity to antagonize fibroblast growth factor-induced extracellular signal-regulated kinase activation. Our results imply that Mnk1-mediated serine phosphorylation of hSpry2 constitutes a regulatory mechanism to extend the temporal range of Spry activity.     

Regulation of ion channels by protein tyrosine phosphorylation

Ion channels are regulated by protein phosphorylation and dephosphorylation of serine, threonine, and tyrosine residues. Evidence for the latter process, tyrosine phosphorylation, has increased substantially since this topic was last reviewed. In this review, we present a comprehensive summary and synthesis of the literature regarding the mechanism and function of ion channel regulation by protein tyrosine kinases and phosphatases. Coverage includes the majority of voltage-gated, ligand-gated, and second messenger-gated channels as well as several types of channels that have not yet been cloned, including store-operated Ca2+ channels, nonselective cation channels, and epithelial Na+ and Cl- channels. Additionally, we discuss the critical roles that channel-associated scaffolding proteins may play in localizing protein tyrosine kinases and phosphatases to the vicinity of ion channels.     

Regulation of flagellar glycoprotein movements by protein phosphorylation

Cross-linking of surface exposed domains on certain Chlamydomonas flagellar membrane glycoproteins induces their movement within the plane of the flagellar membrane. A number of observations suggest that active movements of flagellar membrane glycoproteins are associated with the processes of whole cell gliding motility and the early events of fertilization in Chlamydomonas. Protein redistribution is totally inhibited if the free calcium concentration in the medium is 10(-7) M or below or in the presence of a number of calcium channel blockers (Bloodgood, R. A., N. L. Salomonsky, J. Cell Sci. 96, 27-33 (1990]. The present report demonstrates that glycoprotein redistribution in vivo is inhibited reversibly by three different protein kinase inhibitors: H-7, H-8 and staurosporine. Taken together, these observations suggest that the flagellum uses a signaling pathway that involves calcium influx induced by glycoprotein cross-linking, calcium activation of a protein kinase and specific protein phosphorylation to initiate flagellar surface dynamics.     

Regulation of nicotinic acetylcholine receptors by protein phosphorylation

Neurotransmitter receptors and ion channels play a critical role in the transduction of signals at chemical synapses. The modulation of neurotransmitter receptor and ion channel function by protein phosphorylation is one of the major regulatory mechanisms in the control of synaptic transmission. The nicotinic acetylcholine receptor (nAcChR) has provided an excellent model system in which to study the modulation of neurotransmitter receptors and ion channels by protein phosphorylation since the structure and function of this receptor have been so extensively characterized. In this article, the structure of the nAcChR from the electric organ of electric fish, skeletal muscle, and the central and peripheral nervous system will be briefly reviewed. Emphasis will be placed on the regulation of the phosphorylation of nAcChR by second messengers and by neurotransmitters and hormones. In addition, recent studies on the functional modulation of nicotinic receptors by protein phosphorylation will be reviewed.     

Regulation of alternative splicing by reversible protein phosphorylation

The vast majority of human protein-coding genes are subject to alternative splicing, which allows the generation of more than one protein isoform from a single gene. Cells can change alternative splicing patterns in response to a signal, which creates protein variants with different biological properties. The selection of alternative splice sites is governed by the dynamic formation of protein complexes on the processed pre-mRNA. A unique set of these splicing regulatory proteins assembles on different pre-mRNAs, generating a "splicing" or "messenger ribonucleoprotein code" that determines exon recognition. By influencing protein/protein and protein/RNA interactions, reversible protein phosphorylation modulates the assembly of regulatory proteins on pre-mRNA and therefore contributes to the splicing code. Studies of the serine/arginine-rich protein class of regulators identified different kinases and protein phosphatase 1 as the molecules that control reversible phosphorylation, which controls not only splice site selection, but also the localization of serine/arginine-rich proteins and mRNA export. The involvement of protein phosphatase 1 explains why second messengers like cAMP and ceramide that control the activity of this phosphatase influence alternative splicing. The emerging mechanistic links between splicing regulatory proteins and known signal transduction pathways now allow in detail the understanding how cellular signals modulate gene expression by influencing alternative splicing. This knowledge can be applied to human diseases that are caused by the selection of wrong splice sites.     

Regulation of protein phosphorylation by polyamines in hepatocytes

The effects of natural aliphatic polyamines on basal and hormone-stimulated protein phosphorylations in hepatocytes were studied. Cells isolated from adult rats were incubated in suspension with [³²P]orthophosphate, in the absence or presence of polyamines at varying concentrations and for different times; hepatocytes were then exposed to various hormones for 10 min. Phosphoproteins contained in total cell lysates were analyzed by one- and two-dimensional gel electrophoresis and autoradiography. Spermine, the most effective amine, decreased the basal level of phosphorylation of proteins with 46, 34 and 22 kDa, and increased that of a 18 kDa protein. These effects, maximal with an external concentration of 7.5–10 mM, were detectable after a lag period of about 10 min and reached a plateau after 45 min. Prereatment of cells with the polyamine almost completely prevented stimulation of the phosphorylation of the 46 and 34 kDa proteins by insulin; in contrast, the effects of phenylephrine on the same proteins were only partly inhibited, whereas those of glucagon appeared largely unaffected. The major polyamine effect observed in intact cells (i.e., decreased phophorylation) could be reproduced in a cell-free system where no kinase activity persisted. Indeed, spermine added directly to cell extracts strongly accelerated dephosphorylation of the 46 kDa protein and also of the 61 kDa protein identified as pyruvate kinase; furthermore, restoration of the activity of this enzyme occurred concomitantly with dephosphorylation of the 61 kDa protein in the presence of spermine.     

Regulation of monocyte apoptosis by the protein kinase Cdelta-dependent phosphorylation of caspase-3

Monocytes are central components of the innate immune response and normally circulate for a short period of time before undergoing spontaneous apoptosis. During inflammation, differentiation, or oncogenic transformation, the life span of monocytes is prolonged by preventing the activation of the apoptotic program. Here we showed that caspase-3, a cysteine protease required for monocyte apoptosis, is a phosphoprotein. We identified protein kinase Cdelta (PKCdelta) as a member of the protein kinase C family that associates with and phosphorylates caspase-3. The PKCdelta-dependent phosphorylation of caspase-3 resulted in an increase in the activity of caspase-3. This effect of PKCdelta is specific to caspase-3, as evidenced by the absence of similar effects on caspase-9. The activity of PKCdelta precedes the activation of caspase-3 during spontaneous monocyte apoptosis and in monocyte-induced apoptosis. We found that the overexpression of PKCdelta resulted in an increase of apoptosis, whereas its inhibition blocked caspase-3 activity and decreased apoptosis. Our results provided evidence that the PKCdelta-dependent phosphorylation of caspase-3 provided a novel pro-apoptotic mechanism involved in the regulation of monocyte life span.     

Regulation of neurite outgrowth mediated by localized phosphorylation of protein translational factor eEF2 in growth cones

Nerve growth cones contain mRNA and its translational machinery and thereby synthesize protein locally. The regulatory mechanisms in the growth cone, however, remain largely unknown. We previously found that the calcium entry‐induced increase of phosphorylation of eukaryotic elongation factor‐2 (eEF2), a key component of mRNA translation, within growth cones showed growth arrest of neurites. Because dephosphorylated eEF2 and phosphorylated eEF2 are known to promote and inhibit mRNA translation, respectively, the data led to the hypothesis that eEF2‐mediating mRNA translation may regulate neurite outgrowth. Here, we validated the hypothesis by using a chromophore‐assisted light inactivation (CALI) technique to examine the roles of localized eEF2 and eEF2 kinase (EF2K), a specific calcium calmodulin‐dependent enzyme for eEF2 phosphorylation, in advancing growth cones of cultured chick dorsal root ganglion (DRG) neurons. The phosphorylated eEF2 was weakly distributed in advancing growth cones, whereas eEF2 phosphorylation was increased by extracellular adenosine triphosphate (ATP)‐evoked calcium transient through P2 purinoceptors in growth cones and resulted in growth arrest of neurites. The increase of eEF2 phosphorylation within growth cones by inhibition of protein phosphatase 2A known to dephosphorylate eEF2 also showed growth arrest of neurites. CALI of eEF2 within growth cones resulted in retardation of neurite outgrowth, whereas CALI of EF2K enhanced neurite outgrowth temporally. Moreover, CALI of EF2K abolished the ATP‐induced retardation of neurite outgrowth. These findings suggest that an eEF2 phosphorylation state localized to the growth cone regulates neurite outgrowth. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Multisite phosphorylation by Cdk2 and GSK3 controls cyclin E degradation

Autophosphorylation-triggered ubiquitination has been proposed to be the major pathway regulating cyclin E protein abundance: phosphorylation of cyclin E on T380 by its associated CDK allows binding to the receptor subunit, Fbw7, of the SCFFbw7 ubiquitin ligase. We have tested this model in vivo and found it to be an inadequate representation of the pathways that regulate cyclin E degradation. We show that assembly of cyclin E into cyclin E-Cdk2 complexes is required in vivo for turnover by the Fbw7 pathway; that Cdk2 activity is required for cyclin E turnover in vivo because it phosphorylates S384; that phosphorylation of T380 in vivo does not require Cdk2 and is mediated primarily by GSK3; and that two additional phosphorylation sites, T62 and S372, are also required for turnover. Thus, cyclin E turnover is controlled by multiple biological inputs and cannot be understood in terms of autophosphorylation alone.     

Chemical dissection of the APC Repeat 3 multistep phosphorylation by the concerted action of protein kinases CK1 and GSK3

A crucial event in machinery controlled by Wnt signaling is the association of beta-catenin with the adenomatous polyposis coli (APC) protein, which is essential for the degradation of beta-catenin and requires the multiple phosphorylation of APC at six serines (1501, 1503, 1504, 1505, 1507, and 1510) within its repeat three (R3) region. Such a phosphorylation is believed to occur by the concerted action of two protein kinases, CK1 and GSK3, but its mechanistic aspects are a matter of conjecture. Here, by combining the usage of variably phosphorylated peptides reproducing the APC R3 region and Edman degradation assisted localization of residues phosphorylated by individual kinases, we show that the process is initiated by CK1, able to phosphorylate S1510 and S1505, both specified by non-canonical determinants. Phosphorylation of S1505 primes subsequent phosphorylation of S1501 by GSK3. In turn, phospho-S1501 triggers the hierarchical phosphorylation of S1504 and S1507 by CK1. Once phosphorylated, S1507 primes the phosphorylation of both S1510 and S1503 by CK1 and GSK3, respectively, thus completing all six phosphorylation steps. Our data also rule out the intervention of CK2 despite the presence of a potential CK2 phosphoacceptor site, S1510LDE, in the R3 repeat. S1510 is entirely unaffected by CK2, while it is readily phosphorylated even in the unprimed peptide by CK1delta but not by CK1gamma. This discloses a novel motif significantly different from non-canonical sequences phosphorylated by CK1 in other proteins, which appears to be specifically recognized by the delta isoform of CK1.     

Regulation of the polarity of protein trafficking by phosphorylation

The asymmetry of environmental stimuli and the execution of developmental programs at the organism level require a corresponding polarity at the cellular level, in both unicellular and multicellular organisms. In plants, cell polarity is important in major developmental processes such as cell division, cell enlargement, cell morphogenesis, embryogenesis, axis formation, organ development, and defense. One of the most important factors controlling cell polarity is the asymmetric distribution of polarity determinants. In particular, phosphorylation is implicated in the polar distribution of the determinant protein factors, a mechanism conserved in both prokaryotes and eukaryotes. In plants, formation of local gradients of auxin, the morphogenic hormone, is critical for plant developmental processes exhibiting polarity. The auxin efflux carriers PIN-FORMEDs (PINs) localize asymmetrically in the plasma membrane and cause the formation of local auxin gradients throughout the plant. The asymmetry of PIN distribution in the plasma membrane is determined by phosphorylationmediated polar trafficking of PIN proteins. This review discusses recent studies on the role of phosphorylation in polar PIN trafficking.     

Phosphorylation of NBR1 by GSK3 modulates protein aggregation

The autophagy receptor NBR1 (neighbor of BRCA1 gene 1) binds UB/ubiquitin and the autophagosome-conjugated MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) proteins, thereby ensuring ubiquitinated protein degradation. Numerous neurodegenerative and neuromuscular diseases are associated with inappropriate aggregation of ubiquitinated proteins and GSK3 (glycogen synthase kinase 3) activity is involved in several of these proteinopathies. Here we show that NBR1 is a substrate of GSK3. NBR1 phosphorylation by GSK3 at Thr586 prevents the aggregation of ubiquitinated proteins and their selective autophagic degradation. Indeed, NBR1 phosphorylation decreases protein aggregation induced by puromycin or by the DES/desmin N342D mutant found in desminopathy patients and stabilizes ubiquitinated proteins. Importantly, decrease of protein aggregates is due to an inhibition of their formation and not to their autophagic degradation as confirmed by data on Atg7 knockout mice. The relevance of NBR1 phosphorylation in human pathology was investigated. Analysis of muscle biopsies of sporadic inclusion body myositis (sIBM) patients revealed a strong decrease of NBR1 phosphorylation in muscles of sIBM patients that directly correlated with the severity of protein aggregation. We propose that phosphorylation of NBR1 by GSK3 modulates the formation of protein aggregates and that this regulation mechanism is defective in a human muscle proteinopathy.     

Phosphorylation of NBR1 by GSK3 modulates protein aggregation

The autophagy receptor NBR1 (neighbor of BRCA1 gene 1) binds UB/ubiquitin and the autophagosome-conjugated MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) proteins, thereby ensuring ubiquitinated protein degradation. Numerous neurodegenerative and neuromuscular diseases are associated with inappropriate aggregation of ubiquitinated proteins and GSK3 (glycogen synthase kinase 3) activity is involved in several of these proteinopathies. Here we show that NBR1 is a substrate of GSK3. NBR1 phosphorylation by GSK3 at Thr586 prevents the aggregation of ubiquitinated proteins and their selective autophagic degradation. Indeed, NBR1 phosphorylation decreases protein aggregation induced by puromycin or by the DES/desmin N342D mutant found in desminopathy patients and stabilizes ubiquitinated proteins. Importantly, decrease of protein aggregates is due to an inhibition of their formation and not to their autophagic degradation as confirmed by data on Atg7 knockout mice. The relevance of NBR1 phosphorylation in human pathology was investigated. Analysis of muscle biopsies of sporadic inclusion body myositis (sIBM) patients revealed a strong decrease of NBR1 phosphorylation in muscles of sIBM patients that directly correlated with the severity of protein aggregation. We propose that phosphorylation of NBR1 by GSK3 modulates the formation of protein aggregates and that this regulation mechanism is defective in a human muscle proteinopathy.