We have developed a method for visualizing phosphorylation of proteins in living cells using a novel fluorescent indicator composed of two green fluorescent protein (GFP) variants joined by the kinase-inducible domain (KID) of the transcription factor cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB). Phosphorylation of KID by the cAMP-dependent protein kinase A (PKA) decreased the fluorescence resonance energy transfer (FRET) among the flanking GFPs. By transfecting COS-7 cells with an expression vector encoding this indicator protein (termed ART for cAMP-responsive tracer), we were able to visualize activation dynamics of PKA in living cells.

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Mitogen-activated protein kinases (MAPKs) are activated in response to various extracellular stimuli, and their activities are regulated by upstream activating kinases and protein phosphatases such as MAPK phosphatases (MKPs). We report the identification and characterization of a novel MKP termed SKRP1 (SAPK pathway-regulating phosphatase 1). It contains an extended active site sequence motif conserved in all MKPs but lacks a Cdc25 homology domain. Immunoblotting analysis revealed that SKRP1 is constitutively expressed, and its transcripts of 4.0 and 1.0 kb were detected in almost tissues examined. SKRP1 was highly specific for c-Jun N-terminal kinase (JNK) in vitro and effectively suppressed the JNK activation in response to tumor necrosis factor ␣ or thapsigargin. Endogenous SKRP1 was present predominantly in the cytoplasm and colocalized with JNK. However, SKRP1 does not bind directly to its target JNK, but co-precipitation of SKRP1 with the MAPK kinase MKK7, a JNK activator, was found in vitro and in vivo. Furthermore, we found that SKRP1 did not interfere with the co-precipitation of MKK7 with JNK. Together, our findings indicate that SKRP1 interacts with its physiological substrate JNK through MKK7, thereby leading to the precise regulation of JNK activity in vivo.Mitogen-activated protein (MAP) 1 kinase pathways are evolutionarily conserved in eukaryotic cells (1-5). The pathways are essential for physiological processes, such as embryonic development and immune response (3, 4, 6, 7), and regulate cell survival, apoptosis, proliferation, differentiation, and migration (3, 6 -8).In mammals, three major classes of MAP kinases (MAPKs) have been identified, which differ in their substrate specificity and regulation (1)(2)(3)(4)(5)(6)8). These subgroups compose the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs) (7), and the p38/RK/CSBP kinases. ERKs are activated by a range of stimuli including growth factors, cell adhesion, tumor-promoting phorbol esters, and oncogenes (1, 2), whereas JNK and p38 are preferentially activated by proinflammatory cytokines, and a variety of environmental stresses such as UV and osmotic stress (1,3,6,8). For this reason, the latter are classified as stress-activated protein kinases (SAPKs).Activation of the MAPKs is achieved by dual phosphorylation on threonine and tyrosine residues within a Thr-Xaa-Tyr motif located in the kinase subdomain VIII. This phosphorylation is mediated by a dual specificity protein kinase, MAPK kinase (MAPKK), and MAPKK is in turn activated by phosphorylation mediated by a serine/threonine protein kinase, MAPKK kinase (1-3, 5, 8). In addition to these activating kinases, several types of protein phosphatases have been also shown to control MAPK pathways by dephosphorylating the MAPKs or their upstream kinases. These protein phosphatases include tyrosinespecific phosphatases (9, 10), serine/threonine-specific phosphatases (11, 12), and dual specificity phosphatases (DSPs) (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(2...

Stress-activated protein kinase (SAPK) pathway-regulating phosphatase 1 (SKRP1) has been identified as a member of the mitogen-activated protein kinase (MAPK) phosphatase (MKP) family that interacts physically with the MAPK kinase (MAPKK) MKK7, a c-Jun N-terminal kinase (JNK) activator, and inactivates the MAPK JNK pathway. Although these findings indicated that SKRP1 contributes to the precise regulation of JNK signaling, it remains to be elucidated how SKRP1 is integrated into this pathway. We report that SKRP1 also plays a scaffold role for the JNK signaling, judged by the following observations. SKRP1 selectively formed the stable complexes with MKK7 but not with MKK4 and biphasically regulated the MKK7 activity and MKK7-induced gene transcription in vivo. Co-precipitation analysis between SKRP1 and MKK7-activating MAPKK kinases (MAPKKKs) revealed that SKRP1 also interacted with the MAPKKK, apoptosis signal-regulating kinase 1 (ASK1), but not with MAP kinase kinase kinase 1 (MEKK1). Consistent with these findings, SKRP1 expression increased the ASK1-MKK7 complexes in a dose-dependent manner and specifically enhanced the activation of MKK7 by ASK1. Thus, our findings are, to our knowledge, the first evidence to show that an MKP also functions as a scaffold protein for the particular MAPK signaling.

Mitosis utilizes a number of kinesin-related proteins (KRPs). Here we report the identification of a novel KRP termed KRMP1, which has a deduced 1780-amino acid sequence composed of ternary domains. The amino-terminal head domain is most similar to the kinesin motor domain of the MKLP-1 subfamily and has an intrinsic ATPase activity that is diminished by substituting the consensus Lys-168 with Arg. The central stalk domain is predicted to form a long ␣-helical coiled-coil, and can interact with each other in vivo. An in vivo labeling experiment revealed that KRMP1 is phosphorylated, and we also found that the region within the tail domain containing Thr-1604 as the cdc2 kinase phosphorylation site differs from the bimC box conserved in the bimC subfamily of KRPs. Immunofluorescence analysis showed that endogenous KRMP1 was localized predominantly to the cytoplasm during interphase and dispersed throughout the cell during mitosis. Consistent with this finding, overexpressed KRMP1 was detected in a complicated nuclear or cytoplasmic pattern reflecting multiple nuclear localization/export signals. Furthermore, KRMP1 interacted with the mitotic peptidylprolyl isomerase Pin1 in vivo, and an in vitro interaction was detected between the tail domain of KRMP1 and the WW domain of Pin1. Overexpression of KRMP1 caused COS-7 cells to arrest at G 2 -M, and co-expression of Pin1 reversed this effect, indicating their physiological interaction. Together, our results suggest that KRMP1 is a mitotic target regulated by Pin1 and vice versa.Eukaryotic cells have a set of nucleotide-dependent molecular motors to fulfill specific demands on their viability. Cell division is the most essential and evolved system that engages a number of dynein and kinesin-related proteins (KRPs) 1 to move mitotic spindles and chromosomes in a precise and regulated order. Most of the KRPs identified thus far have been shown to be involved in the mitotic process (reviewed in Refs.