Overexpression of atypical protein kinase C in HeLa cells facilitates macropinocytosis via Src activation

Abstract

Atypical protein kinase C (aPKC) is the first recognized kinase oncogene. However, the specific contribution of aPKC to cancer progression is unclear. The pseudosubstrate domain of aPKC is different from the other PKC family members, and therefore a synthetic peptide corresponding to the aPKC pseudosubstrate (aPKC-PS) sequence, which specifically blocks aPKC kinase activity, is a valuable tool to assess the role of aPKC in various cellular processes. Here, we learned that HeLa cells incubated with membrane permeable aPKC-PS peptide displayed dilated heterogeneous vesicles labeled with peptide that were subsequently identified as macropinosomes. A quantitative membrane binding assay revealed that aPKC-PS peptide stimulated aPKC recruitment to membranes and activated Src. Similarly, aPKC overexpression in transfected HeLa cells activated Src and induced macropinosome formation. Src-aPKC interaction was essential; substitution of the proline residues in aPKC that associate with the Src-SH3 binding domain rendered the mutant kinase unable to induce macropinocytosis in transfected cells. We propose that aPKC overexpression is a contributing factor to cell transformation by interacting with and consequently promoting Src activation and constitutive macropinocytosis, which increases uptake of extracellular factors, required for altered cell growth and accelerated cell migration.

Differential signaling during macropinocytosis in response to M-CSF and PMA in macrophages

“…Two recent papers indicate roles of PKCs in macropinosome formation. A fluorescent-protein chimera of the cPKC PKCγ was recruited to PMA-induced macropinosomes in HeLa cells (Yamamoto et al, 2014 ), and overexpression of the aPKC PKCι (PKCλ in mice induced macropinosomes in HeLa cells (Tisdale et al, 2014 ). Additional studies will be required to establish the full range of PKCs which mediate macropinocytosis.…”

“…The cPKC PKCα is recruited to M-CSF-induced macropinocytic cups (Welliver and Swanson, 2012 ). Other PKC isozymes implicated in macropinocytosis are the cPKC PKCγ (Yamamoto et al, 2014 ) and the aPKC PKCι (PKCλ in mice) (Tisdale et al, 2014 ). Rottlerin, which has been used as a PKCδ-specific inhibitor, is a selective inhibitor of constitutive macropinocytosis (Sarkar et al, 2005 ; Fenyvesi et al, 2014 ), virus-related macropinocytosis (Mercer and Helenius, 2009 ; Raghu et al, 2009 ; Sandgren et al, 2010 ; Haspot et al, 2012 ) and parasite-related macropinocytosis (Barrias et al, 2012 ).…”

GAPDH binds Akt to facilitate cargo transport in the early secretory pathway

“…Based on our previous observations that Rab2 stimulates GAPDH binding to membranes, we employed a quantitative membrane binding assay to learn if Akt was co-recruited [29,34]. For this assay, salt-washed HeLa cell membranes were incubated with GAPDH-depleted rat liver cytosol [29] and GTPγS without or with purified recombinant GAPDH (Fig.…”

“…HeLa membranes (~30 μg of total protein) prepared as previously described [4,5] were added to a reaction mixture that contained 27.5 mM Hepes (pH 7.4), 2.75 mM MgOAc, 65 mM KOAc, 5 mM EGTA, 1.8 mM CaCl 2 , 1 mM ATP, 5 mM creatine phosphate, 0.2 IU rabbit muscle creatine phosphokinase, rat liver cytosol (~25 μg total protein, depleted of GAPDH [29]), 1 μm GST-GDP Dissociation Inhibitor-1 or −2 [33], and 2.0 μm GTPγS [32,34]. Purified recombinant GAPDH or GAPDH (Pro 234 Ser) prepared as described above, was added at the concentrations indicated under “Results,” and the reaction mix incubated at 32 °C for the specified times in the presence or absence of purified recombinant in vitro isoprenylated Rab1 or Rab2 [16,29].…”

An albumin‑binding domain and targeting peptide‑based recombinant protein and its enediyne‑integrated analogue exhibit directional delivery and potent inhibitory activity on pancreatic cancer with K‑ras mutation

“…Macropinocytosis is a highly conservative endocytic pathway observed in certain macrophages (33), dendritic (34) and tumor (35) cells that implements transmembrane transport of extracellular substances by the macropinocytosomes formed of plasma membrane folds. Macropinocytosis is significantly enhanced in tumor cells carrying EGFR or proto-oncogene mutations, such as Ras (36), Rac (37) and Sre (38). As reported by Commisso et al (20), macropinocytosis serves an important role in the nutrient uptake of Ras mutant tumor cells, which enables HSA to be transported into tumor cells and further be decomposed into essential amino acids for sustained cell growth and proliferation, suggesting that HSA may be used as a macropinocytosis-targeted drug delivery carrier.…”