Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and coimmunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.

The Phox and Bem1p (PB1) domain constitutes a recently recognized protein-protein interaction domain found in the atypical protein kinase C (aPKC) isoenzymes, /-and PKC; members of mitogen-activated protein kinase (MAPK) modules like MEK5, MEKK2, and MEKK3; and in several scaffold proteins involved in cellular signaling. Among the last group, p62 and Par6 (partitioning-defective 6) are involved in coupling the aPKCs to signaling pathways involved in cell survival, growth control, and cell polarity. By mutation analyses and molecular modeling, we have identified critical residues at the interaction surfaces of the PB1 domains of aPKCs and p62. A basic charge cluster interacts with an acidic loop and helix both in p62 oligomerization and in the aPKC-p62 interaction. Subsequently, we determined the abilities of mammalian PB1 domain proteins to form heteromeric and homomeric complexes mediated by this domain. We report several novel interactions within this family. An interaction between the cell polarity scaffold protein Par6 and MEK5 was found. Furthermore, p62 interacts both with MEK5 and NBR1 in addition to the aPKCs. Evidence for involvement of p62 in MEK5-ERK5 signaling is presented.

MAPK-activated protein kinase 5 (MK5) was recently identified as a physiological substrate of the atypical MAPK ERK3.Complex formation between ERK3 and MK5 results in phosphorylation and activation of MK5, concomitant stabilization of ERK3, and the nuclear exclusion of both proteins. However, ablation of ERK3 in HeLa cells using small interfering RNA or in fibroblasts derived from ERK3 null mice reduces the activity of endogenous MK5 by only 50%, suggesting additional mechanisms of MK5 regulation. Here we identify the ERK3-related kinase ERK4 as a bona fide interaction partner of MK5. Binding of ERK4 to MK5 is accompanied by phosphorylation and activation of MK5. Furthermore, complex formation also results in the relocalization of MK5 from nucleus to cytoplasm. However unlike ERK3, ERK4 is a stable protein, and its half-life is not modified by the presence or absence of MK5. Finally, although knock-down of ERK4 protein in HeLa cells reduces endogenous MK5 activity by ϳ50%, a combination of small interfering RNAs targeting both ERK4 and ERK3 causes a further reduction in the MK5 activity by more than 80%. We conclude that MK5 activation is dependent on both ERK3 and ERK4 in these cells and that these atypical MAPKs are both physiological regulators of MK5 activity.

The atypical protein kinase C (PKC) isoenzymes, /-and PKC, play important roles in cellular signaling pathways regulating proliferation, differentiation, and cell survival. By using green fluorescent protein (GFP) fusion proteins, we found that wild-type PKC localized predominantly to the cytoplasm, whereas both a kinasedefective mutant and an activation loop mutant accumulated in the nucleus. We have mapped a functional nuclear localization signal (NLS) to the N-terminal part of the zinc finger domain of PKC. Leptomycin B treatment induced rapid nuclear accumulation of GFP-as well as endogenous PKC suggesting the existence of a CRM1-dependent nuclear export signal (NES). Consequently, we identified a functional leucine-rich NES in the linker region between the zinc finger and the catalytic domain of PKC. The presence of both the NLS and NES enables a continuous shuttling of PKC between the cytoplasm and nucleus. Our results suggest that the exposure of the NLS in both -and PKC is regulated by intramolecular interactions between the N-terminal part, including the pseudosubstrate sequence, and the catalytic domain. Thus, either deletion of the N-terminal region, including the pseudosubstrate sequence, or a point mutation in this sequence leads to nuclear accumulation of PKC. The ability of the two atypical PKC isoforms to enter the nucleus in HeLa cells upon leptomycin B treatment differs substantially. Although PKC is able to enter the nucleus very rapidly, PKC is much less efficiently imported into the nucleus. This difference can be explained by the different relative strengths of the NLS and NES in PKC compared with PKC.

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