Carol D. Katayama

A semisynthetic epitope for kinase substrates

The ubiquitous nature of protein phosphorylation makes it challenging to map kinase-substrate relationships, which is a necessary step toward defining signaling network architecture. To trace the activity of individual kinases, we developed a semisynthetic reaction scheme, which results in the affinity tagging of substrates of the kinase in question. First, a kinase, engineered to use a bio-orthogonal ATPγS analog, catalyzes thiophosphorylation of its direct substrates. Second, alkylation of thiophosphorylated serine, threonine or tyrosine residues creates an epitope for thiophosphate ester–specific antibodies. We demonstrated the generality of semisynthetic epitope construction with 13 diverse kinases: JNK1, p38α MAPK, Erk1, Erk2, Akt1, PKCδ, PKCε, Cdk1/cyclinB, CK1, Cdc5, GSK3β, Src and Abl. Application of this approach, in cells isolated from a mouse that expressed endogenous levels of an analog-specific (AS) kinase (Erk2), allowed purification of a direct Erk2 substrate.NOTE: In the version of this article initially published online, a sentence was missing a word and did not make sense. The corrected sentence now reads, “Erk2 was immunoprecipitated from each of these cell lines and assayed with A*TPγS analogs; N6-phenethyl ATPγS was a preferred nucleotide substrate for AS Erk2 and was not accepted by wild-type Erk2 (data not shown).” The error has been corrected for all versions of the article.

Schlafen, a New Family of Growth Regulatory Genes that Affect Thymocyte Development

The Schlafen (Slfn) family of genes are differentially regulated during thymocyte maturation and are preferentially expressed in the lymphoid tissues. Ectopic expression of the prototype member Slfn1 early in the T lineage profoundly alters cell growth and development. In these mice, the DP thymocytes fail to complete maturation, and, depending on the transgene dosage, the number of thymocytes is reduced to 1%-30% of normal. Furthermore, expression of the Schlafen family members in fibroblasts and thymoma cells either retards or ablates cell growth. The conceptual protein sequences deduced for each of the family members have no similarity to characterized proteins and must therefore participate in a heretofore unknown regulatory mechanism guiding both cell growth and T cell development.

Targeted Deletion of Protein Kinase C λ Reveals a Distribution of Functions between the Two Atypical Protein Kinase C Isoforms

Protein kinase C λ (PKCλ) is an atypical member of the PKC family of serine/threonine kinases with high similarity to the other atypical family member, PKCζ. This similarity has made it difficult to determine specific roles for the individual atypical isoforms. Both PKCλ and PKCζ have been implicated in the signal transduction, initiated by mediators of innate immunity, that culminates in the activation of MAPKs and NF-κB. In addition, work from invertebrates shows that atypical PKC molecules play a role in embryo development and cell polarity. To determine the unique functions of PKCλ, mice deficient for PKCλ were generated by gene targeting. The ablation of PKCλ results in abnormalities early in gestation with lethality occurring by embryonic day 9. The role of PKCλ in cytokine-mediated cellular activation was studied by making mouse chimeras from PKCλ-deficient embryonic stem cells and C57BL/6 or Rag2-deficient blastocysts. Cell lines derived from these chimeric animals were then used to dissect the role of PKCλ in cytokine responses. Although the mutant cells exhibited alterations in actin stress fibers and focal adhesions, no other phenotypic differences were noted. Contrary to experiments using dominant interfering forms of PKCλ, mutant cells responded normally to TNF, serum, epidermal growth factor, IL-1, and LPS. In addition, no abnormalities were found in T cell development or T cell activation. These data establish that, in vertebrates, the two disparate functions of atypical PKC molecules have been segregated such that PKCζ mediates signal transduction of the innate immune system and PKCλ is essential for early embryogenesis.

Generation of Mice Deficient for Macrophage Galactose- and N-Acetylgalactosamine-Specific Lectin: Limited Role in Lymphoid and Erythroid Homeostasis and Evidence for Multiple Lectins

ABSTRACT Macrophage receptors function in pattern recognition for the induction of innate immunity, in cellular communication to mediate the regulation of adaptive immune responses, and in the clearance of some glycosylated cells or glycoproteins from the circulation. They also function in homeostasis by initiating the engulfment of apoptotic cells. Evidence has suggested that macrophage receptors function to recognize cells that are destined for programmed cell death but not yet overtly apoptotic. We have examined the function of a macrophage receptor specific for unsialylated glycoproteins, known as the mouse macrophage galactose- and N-acetylgalactosamine-specific lectin (mMGL) (Ii et al., J. Biol. Chem. 265:11295-11298, 1990; Sato et al., J. Biochem. [Tokyo] 111:331-336, 1992; Yamamoto et al., Biochemistry 33:8159-8166, 1994). With targeted disruption, we tested whether mMGL is necessary for macrophage function, controlled thymic development, the loss of activated CD8 T cells, and the turnover of red blood cells. Evidence indicates that mMGL may play a nonessential role in several of these macrophage functions. Experiments are presented that indicate the existence of another galactose- and N-acetylgalactosamine-recognizing lectin distinct from mMGL. This may explain the absence of a strong phenotype in mMGL-deficient mice.

Cutting Edge: Extracellular Signal-Related Kinase Is Not Required for Negative Selection of Developing T Cells

Signals initiated through the TCR during development can result in either survival and differentiation or cell death. High affinity signals that induce death elicit a robust yet transient activation of signaling pathways, including Erk, whereas low affinity ligands, which promote survival, generate a gradual and weaker activation of the same pathways. It was recently demonstrated that Erk localizes to distinct cellular locations in response to high and low affinity ligands. Although a requirement for Erk in positive selection is well established, its role in negative selection is controversial and, thus, the importance of Erk relocalization during development is not understood. In this study, we examined the role of Erk in negative selection using mice that are genetically deficient in both Erk1 and Erk2 in T cells. Results from three different models reveal that thymocyte deletion remains intact in the absence of Erk.