Lymphocyte Phosphatase-Associated Phosphoprotein Is a Substrate of Protein Kinase CK2

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a molecular partner of CD45 phosphatase that plays a key role in the regulation of antigen-specific activation of lymphocytes. The functions of LPAP still remain unknown. We believe that studying LPAP phosphorylation pathways could shed light on its functions. In this work, we studied the phosphorylation of LPAP ectopically expressed in non-lymphoid cells in order to determine the effect of LPAP interaction partners on its phosphorylation. We found that phosphorylation at Ser153 and Ser163 in non-hematopoietic HEK293 cells was conserved, while phosphorylation at Ser99 and Ser172 was almost absent. The pattern of LPAP phosphorylation in K562 erythroid and U937 myeloid cells expressing endogenous CD45 protein was similar to that observed in T and B lymphocytes. We demonstrated for the first time that LPAP is a substrate for protein kinase CK2 that phosphorylates it at Ser153, presumably ensuring LPAP resistance to degradation.     

PKR-Like Endoplasmic Reticulum Kinase Is Necessary for Lipogenic Activation during HCMV Infection

PKR-like endoplasmic reticulum (ER) kinase (PERK) is an ER-associated stress sensor protein which phosphorylates eukaryotic initiation factor 2α (eIF2α) to induce translation attenuation in response to ER stress. PERK is also a regulator of lipogenesis during adipocyte differentiation through activation of the cleavage of sterol regulatory element binding protein 1 (SREBP1), resulting in the upregulation of lipogenic enzymes. Our recent studies have shown that human cytomegalovirus (HCMV) infection in human fibroblasts (HF) induces adipocyte-like lipogenesis through the activation of SREBP1. Here, we report that PERK expression is highly increased in HCMV-infected cells and is necessary for HCMV growth. Depletion of PERK, using short hairpin RNA (shRNA), resulted in attenuation of HCMV growth, inhibition of lipid synthesis and reduction of lipogenic gene expression. Examination of the cleavage of SREBP proteins showed PERK depletion inhibited the cleavage of SREBP1, but not SREBP2, in HCMV-infected cells, suggesting different cleavage regulatory mechanisms for SREBP1 and 2. Further studies showed that the depletion of SREBP1, but not SREBP2, reduced lipid synthesis in HCMV infection, suggesting that activation of SREBP1 is sufficient to induce lipogenesis in HCMV infection. The reduction of lipid synthesis by PERK depletion can be partially restored by expressing a Flag-tagged nuclear form of SREBP1a. Our studies also suggest that the induction of PERK in HCMV-infected cells stimulates SREBP1 cleavage by reducing levels of Insig1 (Insulin inducible gene 1) protein; this occurs independent of the phosphorylation of eIF2α. Introduction of an exogenous Insig1-Myc into HCMV infected cells significantly reduced HCMV growth and lipid synthesis. Our data demonstrate that the induction of PERK during HCMV infection is necessary for full activation of lipogenesis; this effect appears to be mediated by limiting the levels of Insig1 thus freeing SREBP1-SCAP complexes for SREBP1 processing.     

Hydrophobic Interaction between the SH2 Domain and the Kinase Domain Is Required for the Activation of Csk

The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the β3-αC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the β3-αC loop. The mutation of the β3-αC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the β3-αC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain.     

Sgt1 Dimerization Is Negatively Regulated by Protein Kinase CK2-mediated Phosphorylation at Ser361

The kinetochore, which consists of centromere DNA and structural proteins, is essential for proper chromosome segregation in eukaryotes. In budding yeast, Sgt1 and Hsp90 are required for the binding of Skp1 to Ctf13 (a component of the core kinetochore complex CBF3) and therefore for the assembly of CBF3. We have previously shown that Sgt1 dimerization is important for this kinetochore assembly mechanism. In this study, we report that protein kinase CK2 phosphorylates Ser³⁶¹ on Sgt1, and this phosphorylation inhibits Sgt1 dimerization.The kinetochore is a structural protein complex located in the centromeric region of the chromosome coupled to spindle microtubules (1, 2). The kinetochore generates a signal to arrest cells during mitosis when it is not properly attached to microtubules, thereby preventing chromosome missegregation, which can lead to aneuploidy (3, 4). The molecular structure of the kinetochore complex of the budding yeast Saccharomyces cerevisiae has been well characterized; it is composed of more than 70 proteins, many of which are conserved in mammals (2).The centromere DNA in the budding yeast is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEIII (25 bp) is essential for centromere function (7) and is bound to a key component of the centromere, the CBF3 complex. The CBF3 complex contains four proteins, Ndc10, Cep3, Ctf13 (8–15), and Skp1 (14, 15), all essential for viability. Mutations in any of the CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (16, 17). All of the kinetochore proteins, except the CDEI-binding Cbf1 (18–20), localize to the kinetochores in a CBF3-dependent manner (2). Thus, CBF3 is a fundamental kinetochore complex, and its mechanism of assembly is of great interest.We have previously found that Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required to form the active Ctf13-Skp1 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction: the tetratricopeptide repeat (21) and the CHORD protein and Sgt1-specific motif. We and others have found that both domains are important for the interaction of Sgt1 with Hsp90 (23–26), which is required for assembly of the core kinetochore complex. This interaction is an initial step in kinetochore activation (24, 26, 27), which is conserved between yeast and humans (28, 29).We have recently shown that Sgt1 dimerization is important for Sgt1-Skp1 binding and therefore for kinetochore assembly (30). In this study, we have found that protein kinase CK2 phosphorylates Sgt1 at Ser³⁶¹, and this phosphorylation inhibits Sgt1 dimerization. Therefore, CK2 appears to regulate kinetochore assembly negatively in budding yeast.     

Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.     

Requirement of STE50 for Osmostress-Induced Activation of the STE11 Mitogen-Activated Protein Kinase Kinase Kinase in the High-Osmolarity Glycerol Response Pathway

Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 mitogen-activated protein (MAP) kinase cascade, which is composed of three tiers of protein kinases: (i) the SSK2, SSK22, and STE11 MAP kinase kinase kinases (MAPKKKs), (ii) the PBS2 MAPKK, and (iii) the HOG1 MAP kinase. Activation of the MAP kinase cascade is mediated by two upstream mechanisms. The SLN1-YPD1-SSK1 two-component osmosensor activates the SSK2 and SSK22 MAPKKKs by direct interaction of the SSK1 response regulator with these MAPKKKs. The second mechanism of HOG1 MAP kinase activation is independent of the two-component osmosensor and involves the SHO1 transmembrane protein and the STE11 MAPKKK. Only PBS2 and HOG1 are common to the two mechanisms. We conducted an exhaustive mutant screening to identify additional elements required for activation of STE11 by osmotic stress. We found that strains with mutations in the STE50 gene, in combination with ssk2Δ ssk22Δ mutations, were unable to induce HOG1 phosphorylation after osmotic stress. Both two-hybrid analyses and coprecipitation assays demonstrated that the N-terminal domain of STE50 binds strongly to the N-terminal domain of STE11. The binding of STE50 to STE11 is constitutive and is not affected by osmotic stress. Furthermore, the two proteins relocalize similarly after osmotic shock. It was concluded that STE50 fulfills an essential role in the activation of the high-osmolarity glycerol response pathway by acting as an integral subunit of the STE11 MAPKKK.     

The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage

The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser⁴⁶ in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser⁴⁶, and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.     

TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.     

Inflammasome Activation Is Critical to the Protective Immune Response during Chemically Induced Squamous Cell Carcinoma

Chronic inflammation affects most stages of tumorigenesis, including initiation, promotion, malignant differentiation, invasion and metastasis. Inflammasomes have been described as involved with persistent inflammation and are known to exert both pro and antitumour effects. We evaluated the influence of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase (CASP)-1 in the antitumor immune response using a multistage model of squamous cell carcinoma (SCC) development. Absence of ASC and CASP-1 resulted in an earlier incidence and increased number of papilloma. Loss of inflammassome function in mice resulted in decreased presence of natural killer (NK), dendritic (DC), CD4⁺, CD8⁺ and CD45RB⁺ T cells in the tumor lesions as well as in lymph nodes (LN) compared with WT mice. Increased percentage of CD4⁺CD25⁺Foxp3⁺ T cells was associated with association with inflammasome loss of function. Moreover, significant differences were also found with neutrophils and macrophage infiltrating the lesions. Myeloperoxidase (MPO), but not elastase (ELA), activity oscillated among the groups during the SCC development. Levels of proinflammatory cytokines IL-1β, IL-18, Tumor Necrosis Factor (TNF)-α and Interferon (IFN)-γ were decreased in the tumor microenvironment in the absence of inflammasome proteins. These observations suggest a link between inflammasome function and SCC tumorigenesis, indicating an important role for inflammasome activation in the control of SCC development.     

Phosphorylation of the Kinase Homology Domain Is Essential for Activation of the A-Type Natriuretic Peptide Receptor

Natriuretic peptide receptor A (NPR-A) is the biological receptor for atrial natriuretic peptide (ANP). Activation of the NPR-A guanylyl cyclase requires ANP binding to the extracellular domain and ATP binding to a putative site within its cytoplasmic region. The allosteric interaction of ATP with the intracellular kinase homology domain (KHD) is hypothesized to derepress the carboxyl-terminal guanylyl cyclase catalytic domain, resulting in the synthesis of the second messenger, cyclic GMP. Here, we show that phosphorylation of the KHD is essential for receptor activation. Using a combination of phosphopeptide mapping techniques, we have identified six residues within the ATP-binding domain (S497, T500, S502, S506, S510, and T513) which are phosphorylated when NPR-A is expressed in HEK 293 cells. Mutation of any one of these Ser or Thr residues to Ala caused reductions in the receptor phosphorylation state, the number and pattern of phosphopeptides observed in tryptic maps, and ANP-dependent guanylyl cyclase activity. The reductions were not explained by decreases in NPR-A protein levels, as indicated by immunoblot analysis and determinations of cyclase activity in the presence of detergent. Conversion of Ser-497 to Ala resulted in the most dramatic decrease in cyclase activity (~20% of wild-type activity), but conversion to an acidic residue (Glu), which mimics the charge of the phosphoserine moiety, had no effect. Simultaneous mutation of five of the phosphorylation sites to Ala resulted in a dephosphorylated receptor which was unresponsive to hormone and had potent dominant negative inhibitory activity. We conclude that phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A.     

Essential role of IkappaB kinase alpha in the constitutive processing of NF-kappaB2 p100

Processing of NF-kappaB2 precursor protein p100 to generate p52 is tightly controlled, which is important for proper function of NF-kappaB. Accordingly, constitutive processing of p100, caused by the loss of its C-terminal processing inhibitory domain due to nfkappab2 gene rearrangements, is associated with the development of various lymphomas and leukemia. In contrast to the physiological processing of p100 triggered by NF-kappaB-inducing kinase (NIK) and its downstream kinase, IkappaB kinase alpha (IKKalpha), which requires the E3 ligase, beta-transducin repeat-containing protein (beta-TrCP), and occurs only in the cytoplasm, the constitutive processing of p100 is independent of beta-TrCP but rather is regulated by the nuclear shuttling of p100. Here, we show that constitutive processing of p100 also requires IKKalpha, but not IKKbeta (IkappaB kinase beta) or IKKgamma (IkappaB kinase gamma). It seems that NIK is also dispensable for this pathogenic processing of p100. These results demonstrate a general role of IKKalpha in p100 processing under both physiological and pathogenic conditions. Additionally, we find that IKKalpha is not required for the nuclear translocation of p100. Thus, these results also indicate that p100 nuclear translocation is not sufficient for the constitutive processing of p100.     

Integrin-mediated Activation of Focal Adhesion Kinase Is Required for Signaling to Jun NH2-terminal Kinase and Progression through the G1 Phase of the Cell Cycle

The extracellular matrix exerts a stringent control on the proliferation of normal cells, suggesting the existence of a mitogenic signaling pathway activated by integrins, but not significantly by growth factor receptors. Herein, we provide evidence that integrins cause a significant and protracted activation of Jun NH2-terminal kinase (JNK), while several growth factors cause more modest or no activation of this enzyme. Integrin-mediated stimulation of JNK required the association of focal adhesion kinase (FAK) with a Src kinase and p130(CAS), the phosphorylation of p130(CAS), and subsequently, the recruitment of Crk. Ras and PI-3K were not required. FAK-JNK signaling was necessary for proper progression through the G1 phase of the cell cycle. These findings establish a role for FAK in both the activation of JNK and the control of the cell cycle, and identify a physiological stimulus for JNK signaling that is consistent with the role of Jun in both proliferation and transformation.     

Protein Kinase CK2 Regulates the Dimerization of Histone Deacetylase 1 (HDAC1) and HDAC2 during Mitosis

Histone deacetylase 1 (HDAC1) and HDAC2 are components of corepressor complexes that are involved in chromatin remodeling and regulation of gene expression by regulating dynamic protein acetylation. HDAC1 and -2 form homo- and heterodimers, and their activity is dependent upon dimer formation. Phosphorylation of HDAC1 and/or HDAC2 in interphase cells is required for the formation of HDAC corepressor complexes. In this study, we show that during mitosis, HDAC2 and, to a lesser extent, HDAC1 phosphorylation levels dramatically increase. When HDAC1 and -2 are displaced from the chromosome during metaphase, they dissociate from each other, but each enzyme remains in association with components of the HDAC corepressor complexes Sin3, NuRD, and CoREST as homodimers. Enzyme inhibition studies and mutational analyses demonstrated that protein kinase CK2-catalyzed phosphorylation of HDAC1 and -2 is crucial for the dissociation of these two enzymes. These results suggest that corepressor complexes, including HDAC1 or HDAC2 homodimers, might target different cellular proteins during mitosis.