Brain type II calcium and calmodulin-dependent protein kinase: characterization of a brain-region specific isozyme and regulation by autophosphorylation

A variety of molecular approaches have been used to investigate the structural and enzymatic properties of rat brain type ll Ca^(2+) and calmodulin-dependent protein kinase (type ll CaM kinase). This thesis describes the isolation and biochemical characterization of a brain-region specific isozyme of the kinase and also the regulation the kinase activity by autophosphorylation.

The cerebellar isozyme of the type ll CaM kinase was purified and its biochemical properties were compared to the forebrain isozyme. The cerebellar isozyme is a large (500-kDa) multimeric enzyme composed of multiple copies of 50-kDa α subunits and 60/58-kDa β/β’ subunits. The holoenzyme contains approximately 2 α subunits and 8 β subunits. This contrasts to the forebrain isozyme, which is also composed of and β/β'subunits, but they are assembled into a holoenzyme of approximately 9 α subunits and 3 β/β ' subunits. The biochemical and enzymatic properties of the two isozymes are similar. The two isozymes differ in their association with subcellular structures. Approximately 85% of the cerebellar isozyme, but only 50% of the forebrain isozyme, remains associated with the particulate fraction after homogenization under standard conditions. Postsynaptic densities purified from forebrain contain the forebrain isozyme, and the kinase subunits make up about 16% of their total protein. Postsynaptic densities purified from cerebellum contain the cerebellar isozyme, but the kinase subunits make up only 1-2% of their total protein.

The enzymatic activity of both isozymes of the type II CaM kinase is regulated by autophosphorylation in a complex manner. The kinase is initially completely dependent on Ca^(2+)/calmodulin for phosphorylation of exogenous substrates as well as for autophosphorylation. Kinase activity becomes partially Ca^(2+) independent after autophosphorylation in the presence of Ca^(2+)/calmodulin. Phosphorylation of only a few subunits in the dodecameric holoenzyme is sufficient to cause this change, suggesting an allosteric interaction between subunits. At the same time, autophosphorylation itself becomes independent of Ca^(2+) These observations suggest that the kinase may be able to exist in at least two stable states, which differ in their requirements for Ca^(2+)/calmodulin.

The autophosphorylation sites that are involved in the regulation of kinase activity have been identified within the primary structure of the α and β subunits. We used the method of reverse phase-HPLC tryptic phosphopeptide mapping to isolate individual phosphorylation sites. The phosphopeptides were then sequenced by gas phase microsequencing. Phosphorylation of a single homologous threonine residue in the α and β subunits is correlated with the production of the Ca^(2+) -independent activity state of the kinase. In addition we have identified several sites that are phosphorylated only during autophosphorylation in the absence of Ca^(2+)/ calmodulin.

Negative regulation of transcription factors by Srb10 cyclin-dependent kinase

The ubiquitin-dependent proteolytic pathway plays an important role in a broad array of cellular processes, inducting cell cycle control and transcription. Biochemical analysis of the ubiquitination of Sic1, the B-type cyclin-dependent kinase (CDK) inhibitor in budding yeast helped to define a ubiquitin ligase complex named SCF^cdc4 (for Skp1, Cdc53/cullin, F-box protein). We found that besides Sic1, the CDK inhibitor Far1 and the replication initiation protein Cdc6 are also substrates of SCF^cdc4 in vitro. A common feature in the ubiquitination of the cell cycle SCF^cdc4 substrates is that they must be phosphorylated by the major cell cycle CDK, Cdc28. Gcn4, a transcription activator involved in the general control of amino acid biosynthesis, is rapidly degraded in an SCF^cdc4-dependent manner in vivo. We have focused on this substrate to investigate the generality of the SCF^cdc4 pathway. Through biochemical fractionations, we found that the Srb10 CDK phosphorylates Gcn4 and thereby marks it for recognition by SCF^cdc4 ubiquitin ligase. Srb10 is a physiological regulator of Gcn4 stability because both phosphorylation and turnover of Gcn4 are diminished in srb10 mutants. Furthermore, we found that at least two different CDKs, Pho85 and Srb10, conspire to promote the rapid degradation of Gcn4 in vivo. The multistress response transcriptional regulator Msn2 is also a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner upon heat stress-induced translocation into the nucleus. Whereas Msn2 is cytoplasmic in resting wild type cells, its nuclear exclusion is partially compromised in srb10 mutant cells. Srb10 has been shown to repress a subset of genes in vivo, and has been proposed to inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase II. Our results suggest a general theme that Srb10 represses the transcription of specific genes by directly antagonizing the transcriptional activators.

Brf1 post-transcriptionally regulates pluripotency and differentiation responses downstream of Erk MAP Kinase

FGF/Erk MAP Kinase Signaling is a central regulator of mouse embryonic stem cell (mESC) self-renewal, pluripotency and differentiation. However, the mechanistic connection between this signaling pathway activity and the gene circuits stabilizing mESCs in vitro remain unclear. Here we show that FGF signaling post-transcriptionally regulates the mESC transcription factor network by controlling the expression of Brf1 (zfp36l1), an AU-rich element mRNA binding protein. Changes in Brf1 level disrupts the expression of core pluripotency-associated genes and attenuates mESC self-renewal without inducing differentiation. These regulatory effects are mediated by rapid and direct destabilization of Brf1 targets, such as Nanog mRNA. Interestingly, enhancing Brf1 expression does not compromise mESC pluripotency, but does preferentially regulate differentiation to mesendoderm by accelerating the expression of primitive streak markers. Together, these studies demonstrate that FGF signals utilize targeted mRNA degradation by Brf1 to enable rapid post-transcriptional control of gene expression.