A-kinase-anchoring protein AKAP95 is targeted to the nuclear matrix and associates with p68 RNA helicase.

The cell nucleus is structurally and functionally organized by the nuclear matrix. We have examined whether the nuclear cAMP-dependent protein kinase-anchoring protein AKAP95 contains specific signals for targeting to the subnuclear compartment and for interaction with other proteins. AKAP95 was expressed in mammalian cells and found to localize exclusively to the nuclear matrix. Mutational analysis was used to identify determinants for nuclear localization and nuclear matrix targeting of AKAP95. These sites were found to be distinct from previously identified DNA and protein kinase A binding domains. The nuclear matrix-targeting site is unique but conserved among members of the AKAP95 family. Direct binding of AKAP95 to isolated nuclear matrix was demonstrated in situ and found to be dependent on the nuclear matrix-targeting site. Moreover, Far Western blot analysis identified at least three AKAP95-binding proteins in nuclear matrix isolated from rat brain. Yeast two-hybrid cloning identified one binding partner as p68 RNA helicase. The helicase and AKAP95 co-localized in the nuclear matrix of mammalian cells, associated in vitro, and were precipitated as a complex from solubilized cell extracts. The results define novel protein-protein interactions among nuclear matrix proteins and suggest a potential role of AKAP95 as a scaffold for coordinating assembly of hormonally responsive transcription complexes.

Identification of cGMP-dependent protein kinase anchoring proteins (GKAPs).

To promote both efficiency and selectivity, many protein kinases and phosphatases are maintained in specific subcellular microenvironments through their association with anchoring proteins. In this study, we describe a new class of proteins, called GKAPS, that specifically bind the Type II cGMP-dependent protein kinase (PKG). GKAPs were detected in rat aorta, brain, and intestine using a protein overlay technique. The PKG binding proteins were distinct from AKAPs, proteins known to bind the cAMP-dependent protein kinase (PKA). Furthermore, a synthetic peptide that blocks association of PKA with AKAPs did not affect the PKG-GKAP interaction. Deletion mutagenesis was used to map the GKAP binding determinants within PKG to the N-terminal regulatory region. While most GKAPs were tissue-specific, a ubiquitous PKG-binding protein was detected and identified as myosin. Analysis of myosin fragments revealed that PKG binds within Subfragment 2. The results define a novel class of anchoring proteins that may target PKG for specific functional roles.

Reverse transcription-PCR analysis of the regulation of the manganese peroxidase gene family.

Manganese peroxidase (MnP) gene expression in the lignin-degrading fungus Phanerochaete chrysosporium is regulated by nutrient nitrogen levels and by Mn(II), the substrate for the enzyme, as well as by heat shock and other factors. Reverse transcription-PCR (RT-PCR) of total RNA can distinguish the mRNAs of each of the three sequenced P. chrysosporium mnp genes, i.e., mnp1, mnp2, and mnp3. Quantitative RT-PCR demonstrates that each of the three transcripts is present at a similar low basal level in nitrogen-sufficient cultures, with or without Mn, and in nitrogen-limited cultures lacking Mn. However, in 5-day-old, nitrogen-limited, stationary cultures supplemented with 180 microM Mn, the levels of the mnp1 and mnp2 transcripts increased approximately 100- and 1,700-fold, respectively, over basal levels. In contrast, under these conditions, the level of the mnp3 transcript did not increase significantly over the basal level. Quantitative RT-PCR of total RNA extracted from nitrogen-deficient, Mn-supplemented cultures on days 2 through 7 demonstrates that whereas the mnp1 transcript was present at relatively low levels on days 3 through 7, the mnp2 transcript level peaked on day 5 and the mnp3 transcript level peaked on day 3. Comparison of total RNA extracted on day 5 from nitrogen-deficient, Mn-supplemented stationary and agitated cultures indicates that in stationary cultures, mnp2 was the major expressed mnp gene, whereas in large agitated cultures, mnp1 was the major expressed mnp gene.

Truncated-gene reporter system for studying the regulation of manganese peroxidase expression.

The expression of manganese peroxidase (MnP) in nitrogen-limited cultures of Phanerochaete chrysosporium is regulated by Mn, heat shock (HS), and H2O2 at the level of gene transcription. We have constructed a homologous gene reporter system to further examine the regulation of two mnp genes, mnp1 and mnp2, encoding individual MnP isozymes. Internal deletions of 234 and 359 bp were made within the coding regions of the mnp1 and mnp2 genes, respectively. The truncated mnp genes were subcloned into the shuttle vector pOGI18, which includes the Schizophylum commune ade5 gene as a selectable marker, and transformed into a P. chrysosporium Ade1 auxotrophic mutant. Northern-blot analysis of purified Ade+ transformants demonstrated that both of the truncated mnp genes were regulated in a manner similar to the endogenous mnp genes with respect to nitrogen limitation and induction by Mn, HS, and H2O2.