Protein kinase C (PKC)-theta, a member of the ‘novel’ subfamily of PKC isoforms, is of singular importance in transducing signals in T-lymphocytes. Since understanding of regulatory phosphorylation of novel PKCs is fragmentary and inconsistent with findings for ‘classical’ PKC isoforms, we investigated three potential phosphorylation sites on PKC-theta; in the activation loop (Thr538), turn motif (Ser676) and hydrophobic motif (Ser695). Combined evidence from phospho-specific antisera and MS demonstrates phosphorylation at all three sites. Unlike its closest paralogue, PKC-delta, lack of negative charge in the activation loop of PKC-theta results in a profound catalytic defect (>100-fold reduction in the T538A mutant); the high sequence similarity between PKC-theta and -delta assists in the formulation of structural hypotheses to account for this major difference. In contrast with mechanisms proposed for other PKC isoforms, phosphorylation at the other two sites does not reconstitute catalytic activity. Activation loop phosphorylation is critical invivo, since the T538A mutant completely lost its capacity to mediate T-cell receptor-stimulation of nuclear factor κB (NF-κB) activation in Jurkat T-cells. Hydrophobic motif phosphorylation also substantially influences PKC-theta catalytic activity (5-fold reduction in the S695A mutant), but does not impair NF-κB activation in Jurkat T-cells. Its mechanism is independent of secondary effects on activation loop phosphorylation and cannot be explained by thermal instability. Turn motif phosphorylation has a limited effect on kinase activity, but negatively regulates other aspects of PKC-theta function, since the S676A mutant is more efficient than wild-type in inducing NF-κB activation in Jurkat T-cells. These findings expand our understanding of the roles of phosphorylation in novel PKCs, and indicate that PKC-theta is a constitutively competent kinase as a consequence of constitutive phosphorylation of its activation loop.

Phosphopeptide mapping identified a major autophosphorylation site, phospho (p)Thr-219, between the tandem C1 domains of the regulatory fragment in protein kinase C (PKC)h. Confirmation of this identification was derived using (p)Thr-219 antisera that reacted with endogenous PKCh in primary CD3 þ T cells after stimulation with phorbol ester, anti-CD3 or vanadate. The T219A mutation abrogated the capacity of PKCh to mediate NF-jB, NF-AT and interleukin-2 promoter transactivation, and reduced PKCh's ability in Jurkat T cells to phosphorylate endogenous cellular substrates. In particular, the T219A mutation impaired crosstalk of PKCh with Akt/PKBa in NF-jB activation. Yet, this novel (p)Thr-219 site did not affect catalytic activity or second-messenger lipid-binding activity in vitro. Instead, the T219A mutation prevented proper recruitment of PKCh in activated T cells. The PKChT219A mutant defects were largely rescued by addition of a myristoylation signal to force its proper membrane localization. We conclude that autophosphorylation of PKCh at Thr-219 plays an important role in the correct targeting and cellular function of PKCh upon antigen receptor ligation.

We report a case of measles inclusion-body encephalitis (MIBE) occurring in an apparently healthy 21-month-old boy 8.5 months after measles-mumps-rubella vaccination. He had no prior evidence of immune deficiency and no history of measles exposure or clinical disease. During hospitalization, a primary immunodeficiency characterized by a profoundly depressed CD8 cell count and dysgammaglobulinemia was demonstrated. A brain biopsy revealed histopathologic features consistent with MIBE, and measles antigens were detected by immunohistochemical staining. Electron microscopy revealed inclusions characteristic of paramyxovirus nucleocapsids within neurons, oligodendroglia, and astrocytes. The presence of measles virus in the brain tissue was confirmed by reverse transcription polymerase chain reaction. The nucleotide sequence in the nucleoprotein and fusion gene regions was identical to that of the Moraten and Schwarz vaccine strains; the fusion gene differed from known genotype A wild-type viruses.

No abstract

Activation loop phosphorylation plays critical regulatory roles for many kinases. Unlike other protein kinase Cs (PKC), PKC-␦ does not require phosphorylation of its activation loop (Thr-507) for in vitro activity. We investigated the structural basis for this unusual capacity and its relevance to PKC-␦ function in intact cells. Mutational analysis demonstrated that activity without Thr-507 phosphorylation depends on 20 residues N-terminal to the kinase domain and a pair of phenylalanines (Phe-500/Phe-527) unique to PKC-␦ in/near the activation loop. Molecular modeling demonstrated that these elements stabilize the activation loop by forming a hydrophobic chain of interactions from the C-lobe to activation loop to N-terminal (helical) extension. In cells PKC-␦ mediates both apoptosis and transcription regulation. We found that the T507A mutant of the PKC-␦ kinase domain resembled the corresponding wild type in mediating apoptosis in transfected HEK293T cells. But the T507A mutant was completely defective in AP-1 and NF-B reporter assays. A novel assay in which the kinase domain of PKC-␦ and its substrate (a fusion protein of PKC substrate peptide with green fluorescent protein) were co-targeted to lipid rafts revealed a major substrate-selective defect of the T507A mutant in phosphorylating the substrate in cells. In vitro analysis showed strong product inhibition on the T507A mutant with particular substrates whose characteristics suggest it contributes to the substrate selective defect of the PKC-␦ T507A mutant in cells. Thus, activation loop phosphorylation of PKC-␦ may regulate its function in cells in a novel way. Protein kinase C (PKC)2 is a family of 9 genes that can be further divided into classical, novel, and atypical PKCs, depending on their structural characteristics and their requirement for activation (1, 2). Each of them is autoinhibited by an intramolecular interaction of the kinase domain with an N-terminal regulatory domain, whose organization differs between subfamilies. Classic PKCs have C1 and C2 domains that bind diacylglycerol and Ca 2ϩ , respectively, for their activation. Novel PKCs have a C1 domain that binds diacylglycerol, but their C2-like domain does not bind Ca 2ϩ . Atypical PKCs do not have the ability to bind either Ca 2ϩ or diacylglycerol, but are activated by other lipids or small G-proteins. Binding of the regulatory region with appropriate cofactors causes a conformational change that releases the autoinhibition and results in activation.Besides the co-factor-induced conformational change, PKC activity is also regulated by phosphorylation on its kinase domain, most importantly on its activation loop (3, 4). The activation loop is a stretch of 20 -30 amino acids located in the catalytic cleft of the kinase domain of all eukaryotic protein kinases that form part of the substrate peptide binding surface. The activation loop is relatively flexible, and undergoes varied forms of conformation regulation between the active and inactive states (5-7). One of the most common modes of k...

-Crystallin is a taxon-specific crystallin, a major component of the eye lens in elephant shrews (Macroscelidea). Sequence analysis of -crystallin from two genera of elephant shrews and expression of recombinant -crystallin show that the protein is a cytoplasmic (class 1) aldehyde dehydrogenase (ALDH1, EC 1.2.1.3) with activity for the oxidation of retinaldehyde to retinoic acid. Unlike many other mammals, elephant shrews have two ALDH1 genes. One encodes ALDH1/-crystallin which, in addition to its very high expression in lens, is also the predominant form of ALDH1 expressed in other parts of the eye. The second gene encodes a "non-lens" ALDH1 (ALDH1-nl) which is the predominant form expressed in liver. This pattern of tissue preference contrasts with other mammals which make use of the same major ALDH1 transcript in both ocular and non-ocular tissues. Thus the gene recruitment of ALDH1/-crystallin as a structural protein in elephant shrew lenses is associated with its collateral recruitment as the major form of ALDH1 expressed in other parts of the eye.

No abstract

AIM1 (absent in melanoma), a candidate suppressor of malignancy in melanoma, is a nonlens member of the betagamma-crystallin superfamily, which contains six predicted betagamma domains. The first betagamma-crystallin domain of AIM1 (AIM1-g1) diverges most in sequence from the superfamily consensus. To examine its ability to fold and behave like a normal betagamma domain, we cloned AIM1-g1 and overexpressed it in Escherichia coli as a recombinant protein. The recombinant domain was found to be a stable, soluble protein, similar to lens protein gammaBeta-crystallin in secondary structure. The tertiary structure of AIM1-g1 is dominated by the contribution of aromatic amino acids and cysteine. AIM1-g1 undergoes concentration-independent, noncovalent homodimerization with no trace of monomer, similar to a one-domain protein spherulin 3a. Since many betagamma domain proteins bind calcium, we have also investigated the calcium-binding properties of AIM1-g1 by various methods. AIM1-g1 binds the calcium-mimic dye Stains-all, the calcium probe terbium (with K(D) 170 microM), and (45)Ca when blotted on a membrane. AIM1-g1 binds calcium (K(D) 30 microM) with a comparatively higher affinity than bovine lens gamma-crystallin (90 microM). However, calcium binding does not induce significant change in the protein conformation in the near- and far-UV CD and in fluorescence. The AIM1-g1 domain is as stable as domains of betagamma-crystallins (betaB2- or gammaS-crystallins) as monitored by guanidinium chloride unfolding (midpoint of unfolding transition is 1.8 M GdmCl), and the stability of the protein is not altered upon binding calcium as evaluated by equilibrium unfolding. These results show that, despite the sequence variation, AIM1-g1 folds such as a betagamma domain, binds calcium and undergoes dimerization.