The cyclin-dependent kinase 11 interacts with 14-3-3 proteins

Cyclin-dependent kinase 11 isoforms (CDK11) are members of the p34(cdc2) superfamily. They have been shown to play a role in RNA processing and apoptosis. In the present study, we investigate whether CDK11 interacts with 14-3-3 proteins. Our study shows that the putative 14-3-3 binding site (113-RHRSHS-118) within the N-terminal domain of CDK11(p110) is functional. Endogenous CDK11(p110) binds directly to 14-3-3 proteins and phosphorylation of the serine 118 within the RHRSHS motif seems to be required for the binding. Besides, CDK11(p110) is capable of interacting with several different isoforms of 14-3-3 proteins both in vitro and in vivo. The interaction of 14-3-3 gamma with CDK11(p110) occurs throughout the entire cell cycle and reaches maximum at the G2/M phase. Interestingly, 14-3-3 gamma shows strong interaction with N-terminal portion of caspase-cleaved CDK11(p110) (CDK11(p60)) product at 48 h after Fas treatment, which correlates with the maximal cleavage level of CDK11(p110) and the maximum activation level of CDK11 kinase activity during apoptosis. Collectively, these results suggest that CDK11 kinases could be regulated by interaction with 14-3-3 proteins during cell cycle and apoptosis.     

Regulation of Dyrk1A kinase activity by 14-3-3

Dual-specificity tyrosine(Y) regulated kinase 1A (DYRK1A) is a serine/threonine protein kinase implicated in mental retardation resulting from Down syndrome. In this study, we carried out yeast two-hybrid screening to find proteins regulating DYRK1A kinase activity. We identified 14-3-3 as a Dyrk1A interacting protein, which is consistent with the previous finding of the interaction between the yeast orthologues Yak1p and Bmh1/2p. We showed the interaction between Dyrk1A and 14-3-3 in vitro and in vivo. The binding required the N-terminus of Dyrk1A and was independent of the Dyrk1A phosphorylation status. Functionally, 14-3-3 binding increased Dyrk1A kinase activity in a dose dependent manner in vitro. In vivo, a small peptide inhibiting 14-3-3 binding, sc138, decreased Dyrk1A kinase activity in COS7. In summary, these results suggest that DYRK1A kinase activity could be regulated by the interaction of 14-3-3.     

14-3-3 binding to the IGF-1 receptor is mediated by serine autophosphorylation

The phosphoserine-binding 14-3-3 proteins have been implicated in playing a role in mitogenic and apoptotic signaling pathways. Binding of 14-3-3 proteins to phosphoserine residues in the C-terminus of the insulin-like growth factor-1 receptor (IGF-1R) has been described to occur in a variety of cell systems, but the kinase responsible for this serine phosphorylation has not been identified yet. Here we present evidence that the isolated dimeric insulin-like growth factor-1 receptor kinase domain (IGFKD) contains a dual specific (i.e. tyrosine/serine) kinase activity that mediates autophosphorylation of C-terminal serine residues in the enzyme. From the total phosphate incorporation of approximately 4 mol per mol kinase subunit, 1 mol accounts for serine phosphate. However, tyrosine autophosphorylation proceeds more rapidly than autophosphorylation of serine residues (t(1/2) approximately 1 min vs. t(1/2) approximately 5 min). Moreover, dot-blot and far-Western analyses reveal that serine autophosphorylation of IGFKD is sufficient to promote binding of 14-3-3 proteins in vitro. The proof that dual kinase activity of IGFKD is necessary and sufficient for 14-3-3 binding was obtained with an inactive kinase mutant that was phosphorylated on serine residues in a stoichiometric reaction with the catalytically active enzyme. Thus, the IGF-1R itself might be responsible for the serine autophosphorylation which leads to recognition of 14-3-3 proteins in vivo.     

CDK11p110 plays a critical role in the tumorigenicity of esophageal squamous cell carcinoma cells and is a potential drug target

Esophageal squamous cell carcinoma (ESCC) is a serious malignancy with limited options for targeted therapy. The exploration of novel targeted therapies for combating ESCC is urgently needed. Cyclin-dependent kinases (CDKs) play important roles in the progression of cancers; however, the function of CDK11^p110 (cyclin-dependent kinase 11^p110) in ESCC is still unknown. Here, we investigated the effects and molecular mechanisms of CDK11^p110 in the proliferation and growth of ESCC by examining the expression of CDK11^p110 in ESCC tissues and by detecting phenotypic changes in ESCC cells after CDK11^p110 knockdown or overexpression in vitro and in vivo. According to the tissue microarray analysis, compared with its expression level in normal tissues, the expression level of CDK11^p110 was significantly elevated in ESCC tissues; this result was in concordance with the data in TCGA (The Cancer Genome Atlas) datasets. In addition, RNAi-mediated CDK11^p110 silencing exerted a substantial inhibitory effect on the proliferation, clonogenicity and migration ability of ESCC cells. Further study indicated that CDK11^p110 knockdown arrested ESCC cells in the G2/M phase of the cell cycle and induced cell apoptosis. Moreover, stable shRNA-mediated CDK11^p110 knockdown inhibited tumor growth in an ESCC xenograft model, and overexpression of CDK11^p110 enhanced tumor growth. In addition, the Ki67 proliferation index was closely associated with the elevation or depletion of CDK11^p110 in vivo. In summary, this study provides evidence that CDK11^p110 play a critical role in the tumorigenicity of ESCC cells, which suggests that CDK11^p110 may be a promising therapeutic target in ESCC.

Abbreviations: CDKs: cyclin-dependent kinases; CDK11: Cyclin-dependent kinase 11; CDK11^p110: Cyclin-dependent kinase 11^p110, the larger isomer of cyclin-dependent kinase 11; ESCC: esophageal squamous cell carcinoma; FACS: fluorescence-activated cell sorting; FDA: the Food and Drug Administration; TCGA: The Cancer Genome Atlas; TMA: tissue microarray.     

Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-3 proteins

Tyrosine hydroxylase (TH) is phosphorylated by CaM kinase II and is activated in situ in response to a variety of stimuli that increase intracellular Ca(2+). We report here, using baculovirus-expressed TH, that the 14-3-3 protein binds and activates the expressed TH when the enzyme is phosphorylated at Ser-19, a site of CaM kinase II-dependent phosphorylation located in the regulatory domain of TH. Site-directed mutagenesis showed that a TH mutant in which Ser-19 was substituted by Ala retained enzymatic activity at the same level as the non-mutated enzyme, but was a poor substrate for CaM kinase II and did not bind the 14-3-3 protein. Likewise, a synthetic phosphopeptide (FRRAVpSELDA) corresponding to the part of the TH sequence, including phosphoSer-19, inhibited the interaction between the expressed TH and 14-3-3, while the phosphopeptide (GRRQpSLIED) corresponding to the site of cAMP-dependent phosphorylation (Ser-40) had little effect on complex formation. The complex was very stable with a dissociation constant of 3 nM. Furthermore, analysis of PC12nnr5 cells transfected with myc-tagged 14-3-3 showed that 14-3-3 formed a complex with endogenous TH when the cultured cells were exposed to a high K(+) concentration that increases intracellular Ca(2+) and phosphorylation of Ser-19 in TH. These findings suggest that the 14-3-3 protein participates in the stimulus-coupled regulation of catecholamine synthesis that occurs in response to depolarization-evoked, Ca(2+)-dependent phosphorylation of TH.     

Interaction of phosphorylated tyrosine hydroxylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependent association

Tyrosine hydroxylase (TH) has been reported to require binding of 14-3-3 proteins for optimal activation by phosphorylation. We examined the effects of phosphorylation at Ser19, Ser31 and Ser40 of bovine TH and human TH isoforms on their binding to the 14-3-3 proteins BMH1/BMH2, as well as 14-3-3 zeta and a mixture of sheep brain 14-3-3 proteins. Phosphorylation of Ser31 did not result in 14-3-3 binding, however, phosphorylation of TH on Ser40 increased its affinity towards the yeast 14-3-3 isoforms BMH1/BMH2 and sheep brain 14-3-3, but not for 14-3-3 zeta. On phosphorylation of both Ser19 and Ser40, binding to the 14-3-3 zeta isoform also occurred, and the binding affinity to BMH1 and sheep brain 14-3-3 increased. Both phosphoserine-specific antibodies directed against the 10 amino acids surrounding Ser19 or Ser40 of TH, and the phosphorylated peptides themselves, inhibited the association between phosphorylated TH and 14-3-3 proteins. This was also found when heparin was added, or after proteolytic removal of the N-terminal 37 amino acids of Ser40-phosphorylated TH. Binding of BMH1 to phosphorylated TH decreased the rate of dephosphorylation by protein phosphatase 2A, but no significant change in enzymatic activity was observed in the presence of BMH1. These findings further support a role for 14-3-3 proteins in the regulation of catecholamine biosynthesis and demonstrate isoform specificity for both TH and 14-3-3 proteins.     

Protein kinase B (AKT) regulates SYK activity and shuttling through 14-3-3 and importin 7

The Protein kinase B (AKT) regulates a plethora of intracellular signaling proteins to fine-tune signaling of multiple pathways. Here, we found that following B-cell receptor (BCR)-induced tyrosine phosphorylation of the cytoplasmic tyrosine kinase SYK and the adaptor BLNK, the AKT/PKB enzyme strongly induced BLNK (>100-fold) and SYK (>100-fold) serine/threonine phosphorylation (pS/pT). Increased phosphorylation promoted 14-3-3 binding to BLNK (37-fold) and SYK (2.5-fold) in a pS/pT-concentration dependent manner. We also demonstrated that the AKT inhibitor MK2206 reduced pS/pT of both BLNK (3-fold) and SYK (2.5-fold). Notably, the AKT phosphatase, PHLPP2 maintained the activating phosphorylation of BLNK at Y84 and increased protein stability (8.5-fold). In addition, 14-3-3 was required for the regulation SYK⿿s interaction with BLNK and attenuated SYK binding to Importin 7 (5-fold), thereby perturbing shuttling to the nucleus. Moreover, 14-3-3 proteins also sustained tyrosine phosphorylation of SYK and BLNK. Furthermore, substitution of S295 or S297 for alanine abrogated SYK⿿s binding to Importin 7. SYK with S295A or S297A replacements showed intense pY525/526 phosphorylation, and BLNK pY84 phosphorylation correlated with the SYK pY525/526 phosphorylation level. Conversely, the corresponding mutations to aspartic acid in SYK reduced pY525/526 phosphorylation. Collectively, these and previous results suggest that AKT and 14-3-3 proteins down-regulate the activity of several BCR-associated components, including BTK, BLNK and SYK and also inhibit SYK⿿s interaction with Importin 7.     

14-3-3 protein directly interacts with the kinase domain of calcium/calmodulin-dependent protein kinase kinase (CaMKK2)

Background

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca²⁺/calmodulin-dependent kinase (CaMK) family involved in adiposity regulation, glucose homeostasis and cancer. This upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase is inhibited by phosphorylation, which also triggers an association with the scaffolding protein 14-3-3. However, the role of 14-3-3 in the regulation of CaMKK2 remains unknown.

Phosphorylation of mixed lineage kinase MLK3 by cyclin-dependent kinases CDK1 and CDK2 controls ovarian cancer cell division

Mixed lineage kinase 3 (MLK3) is a serine/threonine mitogen-activated protein kinase kinase kinase that promotes the activation of multiple mitogen-activated protein kinase pathways and is required for invasion and proliferation of ovarian cancer cells. Inhibition of MLK activity causes G2/M arrest in HeLa cells; however, the regulation of MLK3 during ovarian cancer cell cycle progression is not known. Here, we found that MLK3 is phosphorylated in mitosis and that inhibition of cyclin-dependent kinase 1 (CDK1) prevented MLK3 phosphorylation. In addition, we observed that c-Jun N-terminal kinase, a downstream target of MLK3 and a direct target of MKK4 (SEK1), was activated in G2 phase when CDK2 activity is increased and then inactivated at the beginning of mitosis concurrent with the increase in CDK1 and MLK3 phosphorylation. Using in vitro kinase assays and phosphomutants, we determined that CDK1 phosphorylates MLK3 on Ser548 and decreases MLK3 activity during mitosis, whereas CDK2 phosphorylates MLK3 on Ser770 and increases MLK3 activity during G1/S and G2 phases. We also found that MLK3 inhibition causes a reduction in cell proliferation and a cell cycle arrest in ovarian cancer cells, suggesting that MLK3 is required for ovarian cancer cell cycle progression. Taken together, our results suggest that phosphorylation of MLK3 by CDK1 and CDK2 is important for the regulation of MLK3 and c-Jun N-terminal kinase activities during G1/S, G2, and M phases in ovarian cancer cell division.     

Constitutively and autonomously active protein kinase C associated with 14-3-3 zeta in the rodent brain

Persistent activation of protein kinase C (PKC) is required for the expression of synaptic plasticity in the brain. There are several mechanisms proposed that can lead to the prolonged activation of PKC. These include long lasting production of lipid activators (diacylglycerol and fatty acid) through mitogen-activated protein (MAP) kinase pathway, and a modification of PKC by reactive oxygen species. In nerve growth factor (NGF)-differentiated PC12 cells, we found that constitutive and autonomous Ca2+-independent PKC activity is associated with 14-3-3 zeta. Because PKC and 14-3-3 zeta are both involved in synaptic plasticity and learning and memory, we examined whether PKC interacts with 14-3-3 zeta in the brain and whether the PKC/14-3-3 zeta complex has autonomous activity. Here we show that three subclasses of PKC, Ca2+-dependent classical PKC, Ca2+-independent novel PKC, and Ca2+-independent and diacylglycerol-insensitive atypical PKC, all interact with 14-3-3 zeta in the rodent brain. The pool size of 14-3-3 zeta bound form of PKC is small (1-4% of each PKC isoform), but they show constitutive and autonomous activity. Our study indicates that the binding of PKC with 14-3-3 zeta is at least in part independent of phosphorylation of PKC and that the C1 domain of PKC is involved in the binding. As both molecules are enriched in synaptic locus, the constitutive PKC activity and its interaction with 14-3-3 zeta could be a mechanism for the persistent PKC activation in the brain.     

Role of 14-3-3 proteins in the regulation of neutral trehalase in the yeast Saccharomyces cerevisiae

In higher eukaryotes, 14-3-3 proteins participate in numerous cellular processes, and carry out their function through a variety of different molecular mechanisms, including regulation of protein localization and enzyme activation. Here, it is shown that the two yeast 14-3-3 homologues, Bmh1p and Bmh2p, form a complex with neutral trehalase (Nth1p), an enzyme that is responsible for trehalose degradation and is required in a variety of stress conditions. In a purified in vitro system, either one of the two 14-3-3 yeast isoforms are necessary for complete activation of neutral trehalase (Nth1p) after phosphorylation by PKA. It is further demonstrated that Bmh1p and Bmh2p bind to the amino-terminal region of phosphorylated trehalase, thereby modulating its enzymatic activity. This work represents the first demonstration of enzyme activation mediated by 14-3-3 binding in yeast.     

Interaction of 14-3-3 proteins with the insulin-like growth factor I receptor (IGFIR): evidence for a role of 14-3-3 proteins in IGFIR signaling

We have extended our previous yeast two-hybrid findings to show that 14-3-3beta also interacts with the insulin-like growth factor I receptor (IGFIR) in mammalian cells overexpressing both proteins and that the interaction involves serine 1283 and is dependent on receptor activation. Treatment of cells with the phorbol ester PMA stimulates the interaction of 14-3-3beta with the IGFIR in the absence of receptor tyrosine phosphorylation, suggesting that receptor activation leads to activation of an endogenous protein kinase that catalyzes the phosphorylation of serine 1283. To investigate the role of 14-3-3 proteins in IGF signal transduction, IGFIR structure-function studies were performed. Mutation of serine 1283 alone (S1283A) (a mutation that decreases but does not abolish the interaction of the IGFIR with 14-3-3) did not affect anchorage-independent growth of NIH 3T3 fibroblasts overexpressing the mutant receptor. However, the simultaneous mutation of this residue and the truncation of the C-terminal 27 residues of the receptor (Delta1310/S1283A) abolished the interaction of the receptor with 14-3-3 and reversed the enhanced colony formation observed with the IGFIR truncation mutation alone (Delta1310). The difference between the Delta1310 and Delta1310/S1283A transfectants in the soft agar assay was confirmed by tumorigenesis experiments. These findings suggest that 14-3-3 proteins interact with the IGFIR in vivo and that this interaction may play a role in a transformation pathway signaled by the IGFIR.     

CDK11p58 protein kinase activity is associated with Bcl-2 down-regulation in pro-apoptosis pathway

CDK11^p58, a G2/M-specific protein kinase, has been shown to be associated with apoptosis in many cell lines, with largely unknown mechanisms. Our previous study proved that CDK11^p58-enhanced cycloheximide (CHX)-induced apoptosis in SMMC-7721 hepatocarcinoma cells. Here we report for the first time that ectopic expression of CDK11^p58 down-regulates Bcl-2 expression and its Ser70, Ser87 phosphorylation in CHX-induced apoptosis in SMMC-7721 cells. Overexpression of Bcl-2 counteracts the pro-apoptotic activity of CDK11^p58. Furthermore, we confirm that the kinase activity of CDK11^p58 is essential to the down-regulation of Bcl-2 as well as apoptosis. Taken together, these results demonstrate that CDK11^p58 down-regulates Bcl-2 in pro-apoptosis pathway depending on its kinase activity, which elicits survival signal in hepatocarcinoma cells.     

14-3-3beta is a p90 ribosomal S6 kinase (RSK) isoform 1-binding protein that negatively regulates RSK kinase activity

p90 ribosomal S6 kinase 1 (RSK1) is a serine/threonine kinase that is activated by extracellular signal-related kinases 1/2 and phosphoinositide-dependent protein kinase 1 upon mitogen stimulation. Under basal conditions, RSK1 is located in the cytosol and upon stimulation, RSK1 translocates to the plasma membrane where it is fully activated. The ability of RSK1 to bind the adapter protein 14-3-3beta was investigated because RSK1 contains several putative 14-3-3-binding motifs. We demonstrate that RSK1 specifically and directly binds 14-3-3beta. This interaction was dependent on phosphorylation of serine 154 within the motif RLSKEV of RSK1. Binding of RSK1 to 14-3-3beta was maximal under basal conditions and decreased significantly upon mitogen stimulation. After 5 min of serum stimulation, a portion of 14-3-3beta and RSK1 translocated to the membrane fraction, and immunofluorescence studies demonstrated colocalization of RSK1 and 14-3-3beta at the plasma membrane in vivo. Incubation of recombinant RSK1 with 14-3-3beta decreased RSK1 kinase activity by approximately 50%. Mutation of RSK1 serine 154 increased both basal and serum-stimulated RSK activity. In addition, the epidermal growth factor response of RSK1S154A was enhanced compared with wild type RSK. The amount of RSK1S154A was significantly increased in the membrane fraction under basal conditions. Increased phosphorylation of two sites essential for RSK1 kinase activity (Ser(380) and Ser(363)) in RSK1S154A compared with RSK1 wild type, demonstrated that 14-3-3 interferes with RSK1 phosphorylation. These data suggest that 14-3-3beta binding negatively regulates RSK1 activity to maintain signal specificity and that association/dissociation of the 14-3-3beta-RSK1 complex is likely to be important for mitogen-mediated RSK1 activation.     

Casein kinase 2 is essential for mitophagy

Mitophagy is a process that selectively degrades mitochondria. When mitophagy is induced in yeast, the mitochondrial outer membrane protein Atg32 is phosphorylated, interacts with the adaptor protein Atg11 and is recruited into the vacuole with mitochondria. We screened kinase‐deleted yeast strains and found that CK2 is essential for Atg32 phosphorylation, Atg32–Atg11 interaction and mitophagy. Inhibition of CK2 specifically blocks mitophagy, but not macroautophagy, pexophagy or the Cvt pathway. In vitro, CK2 phosphorylates Atg32 at serine 114 and serine 119. We conclude that CK2 regulates mitophagy by directly phosphorylating Atg32.     

Engagement of beta2 integrins recruits 14-3-3 proteins to c-Cbl in human neutrophils

We found that engagement of beta2 integrins on human neutrophils triggered both tyrosine and serine phosphorylation of c-Cbl. Pretreatment of the neutrophils with the broad range protein kinase C (PKC) inhibitor GF-109203X blocked the serine but not the tyrosine phosphorylation of c-Cbl. Moreover, the Src kinase inhibitor PP1 prevented the beta2 integrin-induced tyrosine phosphorylation of c-Cbl but not the simultaneous serine phosphorylation. These results indicate that Src family kinases and PKC can separately modulate the properties of c-Cbl. Indeed, tyrosine kinase-dependent phosphorylation of c-Cbl regulated the ubiquitin ligase activity of that protein, whereas PKC-dependent phosphorylation of c-Cbl had no such effect. Instead, c-Cbl that underwent PKC-induced serine phosphorylation associated with the scaffolding and anti-apoptotic 14-3-3 proteins. Consequently, c-Cbl can independently target proteins for degradation or intracellular localization and may initiate an anti-apoptotic signal in neutrophils.